Viral inactivated biological mixture

ABSTRACT

The invention relates to a viral inactivated biological liquid or dry mixture and to its preparation. Principally, the invention relates, but is not limited, to a mixture derived from a platelet source.

FIELD OF THE INVENTION

Generally, the invention relates to a viral inactivated biologicalliquid or dry mixture and to its preparation. Principally, the inventionrelates, but is not limited, to a viral inactivated platelet extract andpreparation thereof.

BACKGROUND OF THE INVENTION

Platelets are small, irregularly-shaped a-nuclear cells that play afundamental role in hemostasis and healing. Platelets contain a completearray of pre-synthesized proteins, among which are signaling proteins,cytoskeletal proteins, membrane proteins and regulatory proteins. Theyare involved in key stages of tissue regeneration and healing processesat the site of injury, mainly due to the content of platelet granulescomprising a multitude of bioactive molecules including growth factors(GFs), cytokines and chemokines.

Platelet growth factors such as platelet-derived growth factor (PDGF),transforming growth factor (TGF), basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF) and others are key players inall the following phases of the wound healing cascade: inflammatory,proliferative and remodeling phase.

Studies have shown that platelet derived growth factors stimulateangiogenesis, mitogenesis, cell proliferation, neutrophils andmacrophages, collagen synthesis, wound contraction, extracellular matrixsynthesis, epithelialization and chemotaxis.

Platelets are routinely used by transfusion e.g. to improve hemostasis.Recently, platelets are increasingly used in the form of Platelet RichPlasma (PRP), also referred to as PRP gel, platelet gel, PRP-clot etc.Typically, PRP is an ex vivo preparation consisting of autologousplatelets concentrated in a limited volume of plasma (Lacci K M, DardikA. Platelet-rich plasma: support for its use in wound healing. Yale JBiol Med. 2010 March; 83(1):1-9).

For topical application, PRP is usually activated by the addition ofthrombin and/or CaCl₂ resulting in the formation of fibrin gel by theinteraction between thrombin (endogenous or exogenous) and fibrinogen.Upon activation, the platelets undergo active degranulation and releasevarious mediators including GFs (Lacci K M, Dardik A, 2010). The use ofPRP for injection currently comprises a small but rapidly growingsegment of the market. The rationale for using PRP in soft and hardtissue augmentation is its potential to enhance tissue regeneration innon-healing injuries, accelerate wound maturity, vascularization andepithelialization, decrease scar formation, and reduce post operativecomplications and morbidity (Lacci K M, Dardik A, 2010).

Studies using activated PRP together with various cell types have shownthat factors e.g. growth factors released from PRP can induce cellproliferation [(e.g. Kanno et al. Platelet-rich plasma enhances humanosteoblast-like cell proliferation and differentiation. J OralMaxillofac Surg. 2005 March; 63(3):362-9; Bertrand-Duchesne et al.Epidermal growth factor released from platelet-rich plasma promotesendothelial cell proliferation in vitro. J Periodontal Res. 2010February; 45(1):87-93; Kakudo et al. Proliferation-promoting effect ofplatelet-rich plasma on human adipose-derived stem cells and humandermal fibroblasts. Plast Reconstr Surg. 2008 November; 122(5):1352-60),modulate the angiogenic capability of human endothelial cells (Sulpiceet al. Cross-talk between the VEGF-A and HGF signalling pathways inendothelial cells. Biol Cell. 2009 September; 101(9):525-39; Rughetti etal. Platelet gel-released supernatant modulates the angiogeniccapability of human endothelial cells. Blood Transfus. 2008 January;6(1):12-7), and induce osteo-inductive properties (Intini G. The use ofplatelet-rich plasma in bone reconstruction therapy. Biomaterials. 2009October; 30(28):4956-66)]. Moreover, activated PRP was found to supportin vitro cell growth and maintained viability of a number of targetcells including myelomas, hybridomas, hepatocytes, fibroblasts andepithelial cells, at a level comparable or superior to the levelsupported by fetal bovine serum (Johansson et al. Platelet lysate: areplacement for fetal bovine serum in animal cell culture?Cytotechnology. 2003 July; 42(2):67-74).

PRP and released growth factors are currently used in various surgicaltissue regeneration procedures, predominantly in orthopedic and dentalsurgery (Nurden et al. Platelets and wound healing. Front Biosci. 2008May 1; 13:3532-48). In orthopedic surgery PRP is used mainly for kneearthroplasty, lumbar spinal fusion, and in intervertebral discdegeneration (reviewed in Nurden et al, 2008). Dentistry andmaxillofacial surgery PRP applications include mainly consolidation oftitanium implants, maxillary sinus augmentation and bone remodeling(reviewed in Nurden et al, 2008). PRP is also increasingly used fortendon and ligament repair, facial plastic and reconstructive surgery,chronic skin wound healing, ophthalmology, facial nerve regeneration, aswell as in cardiac and bariatric surgery (reviewed in Nurden et al,2008).

However, a major disadvantage of the current use of autologous PRP andreleased factors resides in the lack of standardization. Of note,different manual, semi-automated and fully-automated systems forpreparation of PRP are commercially available that differ in parameterssuch as preparation time, platelet yield and collection efficiency(Mazzucco et al. Not every PRP-gel is born equal. Evaluation of growthfactor availability for tissues through four PRP-gel preparations:Fibrinet, RegenPRP-Kit, Plateltex and one manual procedure. Vox Sang.2009 August; 97(2):110-8).

Another important variable is the technique used for platelet activation[autologous, heterologous or recombinant thrombin, calcium chloride orbatroxobin (Rozman P, Bolta Z. Use of platelet growth factors intreating wounds and soft-tissue injuries. Acta Dermatovenerol AlpPanonica Adriat. 2007 December; 16(4):156-65)], which can affect theefficiency of granule release and the amount of secreted GFs (Rozman P,Bolta Z, 2007). Moreover, since platelets are very sensitive tomechanical stress and changes in the surrounding environment, they maybe activated and GFs may be released during processing, prior to theintended activation step (Mazzucco et al, 2009). This uncontrolledactivation may further increase the variability in the composition ofthe final product when using different PRP preparation systems.Additionally, a major inherent weakness of autologous PRP preparation isthat the platelets GFs content varies among individuals, and thereforemay lead to sub-optimal results. Finally, the financial burden ofdedicated machinery, disposable PRP processing kits, and the need fortrained personnel, should be taken into consideration when working withautologous PRP.

Background art includes Su et al. “A virally inactivated functionalgrowth factor preparation from human platelet concentrates”. Vox Sang.2009 August; 97(2):119-128; Burnouf et al. “A novel virally inactivatedhuman platelet lysate preparation rich in TGF-beta, EGF and IGF, anddepleted of PDGF and VEGF”. Biotechnol Appl Biochem. 2010 Aug. 6;56(4):151-60; and U.S. Patent Publication No. US 2012-0156306.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method for preparing aviral-safe biological liquid mixture, the method comprising thefollowing steps: providing a biological liquid mixture; carrying out asolvent detergent (S/D) viral inactivation treatment; contacting the S/Dtreated mixture with an amphiphilic polymer; removing the S/D byhydrophobic interaction chromatography (HIC) and/or by oil extraction;collecting a material comprising a flow through fraction from HIC and/ora liquid fraction from oil extraction; and subjecting the material to atleast one more orthogonal viral inactivation treatment.

In one embodiment of the invention, the amphiphilic polymer isnon-toxic.

Yet, in a further embodiment of the invention, the amphiphilic polymeris a hydrocarbon based surfactant.

Yet, in a further embodiment of the invention, the amphiphilic polymerhas an average molecular weight in the range of about 3.5 to lower thanabout 40 kilodalton.

In one embodiment of the invention, the average molecular weight isabout 30 kilodalton.

Yet, in a further embodiment of the invention, the hydrocarbon basedsurfactant is polyvinylpyrrolidone (PVP).

In one embodiment of the invention, the PVP has an average molecularweight of about 30 kilodalton (kDa).

In one embodiment of the invention, the HIC comprises the steps of:loading the S/D treated and polymer contacted mixture to HIC; washingwith a solution comprising an organic solvent and/or a salt: andcollecting a washed fraction.

In one embodiment of the invention, the organic solvent is ethanol.

In one embodiment of the invention, the salt is NaCl.

In a further embodiment of the invention, the at least one moreorthogonal viral inactivation treatment comprises heat inactivation.

In one embodiment of the invention, the method further comprises a stepof concentrating the material.

In one embodiment of the invention, the method is for preparing aviral-safe platelet extract, and the biological liquid mixture is aplatelet-enriched fraction.

In one embodiment of the invention, the collected material comprises theHIC flow through fraction combined with the HIC washed fraction.

In another aspect, the invention relates to a viral-safe biologicalliquid mixture obtainable according to the method of the invention.

In one embodiment of the invention, the concentration of PVP K25 is inthe range of 0.1% (w/w) to lower than 1% (w/w).

In certain embodiments of the invention, the concentration of PVP K25 isin the range of 0.1% (w/w) to 0.5% (w/w).

In one embodiment of the invention, the PVP K17, K25, K30 concentrationin the viral-safe biological liquid mixture is in the range of 0.01-5%(w/w).

In certain embodiments of the invention, the biological liquid mixtureis a platelet extract enriched with PDGF-AB, PDGF-BB, EGF, VEGF and/orbFGF.

In another aspect, the invention relates to a method for removingsolvent-detergent (S/D) from a biological liquid mixture comprising theS/D, the method comprises the steps of: providing the mixture comprisingthe S/D; contacting the mixture with an amphiphilic polymer; removingthe S/D from the mixture by hydrophobic interaction chromatography (HIC)and/or by oil extraction; and collecting a material comprising a flowthrough fraction from HIC and/or a liquid fraction from oil extraction.

In one embodiment of the invention, the amphiphilic polymer isnon-toxic.

In one embodiment of the invention, the biological liquid mixture is aplatelet-enriched fraction.

In another embodiment of the invention, the mixture compriseschemokines, cytokines, growth factors, trophic factors or a mixturethereof.

In one embodiment of the invention, the HIC comprises the steps of:washing with a solution comprising an organic solvent and/or a salt; andcollecting a washed fraction.

In one embodiment of the invention, the amphiphilic polymer is ahydrocarbon based surfactant.

In one embodiment of the invention, the hydrocarbon based surfactant ispolyvinylpyrrolidone (PVP).

In one embodiment of the invention, the organic solvent is ethanol.

In one embodiment of the invention, the salt is NaCl.

In one embodiment of the invention, the collected material comprises theflow through fraction combined with the wash fraction.

In one embodiment of the methods, the source is contacted first with theS/D and then with the amphiphilic polymer.

In some embodiments of the methods, the PVP concentration in the S/Dtreated source is in the range of about 0.01 to 0.9 mM, 0.01 to 0.3 mM,or 0.025 to 0.3 mM.

In some embodiments of the methods, the HPMC concentration in the S/Dtreated source is in the range of about 0.01 to 0.3 mM.

In some embodiments, the methods further comprise a step of drying thematerial, thereby resulting in a biological dry mixture.

In a certain aspect, it is disclosed a method for preparing a biologicalliquid mixture composition from a biological source. The methodcomprises the following steps: providing the source; providing PVPand/or HPMC; treating the source with a solvent detergent (S/D) to allowviral inactivation and with the PVP and/or HPMC; removing the S/D bycontacting the treated source with a hydrophobic interactionchromatography (HIC) resin; and collecting a material comprising anunbound fraction from HIC.

In a certain aspect, it is disclosed a biological liquid mixturecomposition obtainable according to the disclosed methods.

In one embodiment, the composition comprises a PVP concentration in therange of about 0.07 to 6 mM, 0.07 to 2 mM, or 0.17 to 2 mM.

In another embodiment, the composition comprises a HPMC concentration inthe range of about 0.07 to 1.5 mM.

In a certain aspect, it is disclosed a pharmaceutical compositioncomprising an amphiphilic polymer; a platelet derived protein selectedfrom the group consisting of a chemokine, a growth factor, a cytokine, atrophic factor and a mixture thereof; and a pharmaceutically acceptablecarrier, wherein the amphiphilic polymer is PVP at a concentration inthe range of about 0.07 to 6 mM or HPMC at a concentration in the rangeof about 0.07 to 1.5 mM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows proliferation of 3T3 fibroblast cells treated with aplatelet extract obtained by contacting the lysate with heparin prior toS/D removal (treatment 2 and 4). A platelet extract prepared withoutcontacting the lysate with heparin prior to S/D removal served as thecontrol (treatment 1 and 3).

FIG. 2 shows proliferation of 3T3 fibroblast cells treated with aplatelet extract prepared by contacting the lysate with low molecularweight heparin (LMWH) prior to and during S/D removal (treatment 2). Aplatelet extract obtained following S/D removal in the absence ofcontacting the lysate with low molecular weight heparin (LMWH) prior toS/D removal served as the control (treatment 1).

FIG. 3 shows proliferation of 3T3 fibroblast cells treated with extractsobtained after S/D removal in the presence of different molecular weightPVP polymers: PVP K25—treatment 3; or PVP K30—treatment 7.

FIG. 4 shows proliferation of 3T3 fibroblast cells treated with extractscomprising different concentrations of PVP. Sample 17 (treatment 17) andsample 19 (treatment 19) differ in the second wash of the S/D removal,where 0.1% and 0.5% PVP were used, respectively.

FIG. 5 shows proliferation of 3T3 fibroblast cells treated with largescale platelet extracts obtained after S/D removal in the presence ofPVP K25 (treatment 1) or after S/D removal in the presence of heparin(treatment 2).

In addition, all figures comprise R² fit, median effective concentration(EC50), and 95% Confidence Intervals EC50 values calculated by GraphPadPrism software.

FIG. 6 shows a rat dorsal flap (3×10 cm) at 2 weeks after surgeryperformance. The flap was elevated in cranial to caudal direction. A, B,C, D and E indicate different areas from where samples were taken forhistological analysis. A is closest to the caudal flap attachment andtherefore heals best, whereas E is in the cranial end of the flap, whichshows highest levels of necrosis (dark color). The abdominal andthoracic viscera were removed through ventral midline incision (linealong the center of the flap).

FIG. 7 shows typical staining patterns for normal and healing skin: H&Estaining (epidermal hyperplasia, score 1 and epidermis after completedhealing process, score 0). PCNA staining for dermal and epidermalproliferation (proliferating tissue, score 1 and normal tissue, score 0)and Keratin 6 staining (suprabasal staining, score 1, for healing andbasal staining for regular skin, score 0).

FIG. 8 shows the scores for the adherence grade of the rat dorsal flapafter 2 weeks, as they were tested by gently pulling the flap in thearea A-C (see FIG. 6) away from the wound bed using a tissue forceps.The attachment of the skin flap to the underlying tissue was compared tothe attachment of normal areas of skin and graded 1 to 3 as follows:1=no to low adherence, 2=below but nearly normal adherence or 3=aboutnormal adherence between skin flap and underlying tissue.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

The invention relates to methods for preparing a viral-safe biologicalliquid mixture such as a viral safe platelet extract. Platelets containa complete array of factors involved in key stages of tissueregeneration and healing processes. Currently, whole autologousactivated platelets (derived from the patient) are used for facilitatingwound healing. However, there are multiple disadvantages of using wholeautologous platelets, inter alia, the lack of standardization; thefactors needed for healing may be scarce in the patient's own platelets:the special equipment needed for preparing the mixture of plateletfactors; the procedure is time consuming and requires additional stepswhich are carried out on the patient itself; and the requirement ofmedically trained personnel. These problems can be solved e.g. by usinga platelet extract prepared from multiple donors.

However, human blood-derived products may carry a risk of transmittinginfectious agents such as viruses. Effective reduction of viraltransmission risk can be achieved by including at least two orthogonalviral inactivation steps. Yet, including additional steps in themanufacture of a platelet extract may compromise the recovery andactivity of the factors contained therein.

One of these methods of viral inactivation is “Solvent detergent (S/D)viral inactivation treatment”.

This inactivation includes treatment with S/D and removal of the S/D. Itwas found according to the present invention that the recovery ofcertain growth factors is compromised after S/D removal by HIC.

It was found according to the present invention that recovery of certainplatelet factors e.g. PDGF-AB; PDGF-BB; and bFGF, can be increased bycontacting the S/D treated material prior to and/or during S/D removalwith polyvinylpyrrolidone (PVP) or Hydroxy Propyl Methyl Cellulose(HPMC), which are non-toxic amphiphilic molecules.

It was found that contacting the S/D treated material with PVP and HPMCin accordance to the invention resulted in increased recovery orenrichment of PDGF-AB and other platelet factors e.g. PDGF-BB and bFGF.

These findings are surprising, in view that contacting the S/D treatedmaterial with heparin and low molecular weight heparin (both known tobind certain growth factors) during S/D removal increased the recoveryof factors while contacting S/D treated material with PVP, which is acompletely different compound (having amphiphilic characteristics) had asimilar beneficial effect on growth factor recovery during S/D removal.

Also, the findings are surprising, since addition of PVP K30, K25, K17and K12 under certain tested conditions did not compromise S/D removal.

It was found that using the method according to the invention to removeS/D from a source comprising platelet-derived mixture of factors resultsin high recovery of factors, high biological activity and efficientremoval of S/D.

It was found that, using PVP K12, K17, K30 or PVP K25 during S/D removalincreases recovery of platelets growth/trophic factors. It was foundthat the recovery using K30 was higher than using K25. The materialobtained using PVP K25, and therefore comprising PVP K25, had a higherproliferative activity than material obtained using PVP K30 whichcomprised PVP K30.

The results also show that it is possible to reduce the PVP K25concentration contacted with the platelet factors mixture to below 0.5%or 0.17 mM (thereby decreasing PVP to below 0.5% or 0.17 mM in theextract obtained after S/D removal) and still obtain an increase infactor recovery while maintaining the ability of HIC to efficientlyremove S/D.

The results show that, the presence of different amounts of PVP K25,e.g. 0.1% (0.03 mM) and 0.5% (0.17 mM) in the extract did not affect itsactivity.

The results show that, unlike heparin and dextran sulfate at certainconcentration, the presence of PVP in the final extract did not inhibitthrombin activity. This property of PVP is important especially whenusing fibrin sealant as a delivery agent for the platelet extract(“platelet extract” is one kind of biological liquid mixturecomposition).

The results show that growth factors recovery and activity in thepresence of PVP K25 in large scale process are comparable with those insmall scale.

These results suggest that PVP can be advantageously used during S/Dremoval in order to obtain a final extract having increased biologicalpotency, provided that the type of PVP used and its concentration (e.g.w/w or molarity of PVP in the mixture) does not compromise the S/Dremoval.

In one embodiment, a platelet extract is obtained, after contacting abiological source with PVP in combination with ethanol and NaCl duringan S/D removal step. The extract comprises PDGF-AB/TGF-β1; PDGF-AB/VEGF;TGF-β1/bFGF; and VEGF/bFGF ratios which are similar to the ratios in theWashed Aphaeresis Platelets Leukocyte-Reduced (WAP-LR) starting materialand in the material prior to S/D removal.

These findings paved the way to prepare a biological liquid mixturecomposition according to the invention.

The method of the invention enables to prepare a platelet extract withincreased recovery of cytokines, growth factors, chemokines and/ortrophic factors following removal of S/D.

It is disclosed a method for preparing a viral-safe biological liquidmixture composition from a biological source, the method comprising thefollowing steps: providing the source; providing an amphiphilic polymer,treating the source with a solvent detergent (S/D) to allow viralinactivation and with the amphiphilic polymer; removing the S/D bycontacting the treated source with an hydrophobic interactionchromatography (HIC) resin; and collecting a material comprising anunbound fraction from HIC; wherein the method comprises at least onemore orthogonal viral inactivation treatment, thereby obtaining theviral-safe biological liquid mixture composition.

In one aspect, the invention provides a method for preparing aviral-safe biological liquid mixture, the method comprising thefollowing steps:

providing a source; carrying out a solvent detergent (S/D) viralinactivation treatment; contacting the S/D treated material with a nontoxic amphiphilic polymer, removing the S/D by hydrophobic interactionchromatography (HIC) and/or by oil extraction; and subjecting thematerial to at least one more orthogonal viral inactivation treatment.

Examples of the source include, but are not limited to, body fluids suchas blood; blood fractions, cryoprecipitate, cell cultures, lipophilicproteinaceous agents; cells, cell particles and/or cell organelles; celllysate; platelet lysate; blood buffy coat; animal tissue extracts, suchas bovine lungs, bovine intestines or animal bone extracts gelatin,bovine serum albumin, as well as animal derived water immiscible fats,such as lanoline. The source can be derived from a plurality of donors.

In one aspect it is disclosed a method for removing solvent-detergent(S/D) from a biological source comprising S/D, the method comprises thesteps of: providing the source; providing an amphiphilic polymer;treating the source with S/D and with the amphiphilic polymer; removingthe S/D from the biological source by contacting the treated source withan hydrophobic interaction chromatography (HIC) resin; and collecting amaterial comprising an unbound fraction from HIC.

In one embodiment the method of removing the S/D omits a further step ofoil extraction.

It has been found that the method of the invention can be used to removeS/D in the absence of a step of oil extraction partition.

In one embodiment, the invention relates to a method for preparing aviral-safe platelet extract, the method comprising the following steps:providing a platelet-enriched fraction from more than one donor;carrying out a solvent detergent (S/D) viral inactivation treatment;contacting the S/D treated material with a non toxic amphiphilicpolymer; removing the S/D; and subjecting the material to at least onemore orthogonal viral inactivation treatment.

The term “platelet extract” refers to a biological mixture comprisingplatelet-derived factors. Typically, extracts are cell free.

In one embodiment, the method comprises preparing a platelet lysate. Theterm “lysate” refers to a solution produced when cells are destroyed bydisrupting their cell membranes. Lysis of the platelets and release ofthe factors (e.g. various platelet growth factors and/or trophicfactors) entrapped in the platelets, can be carried out by freezing andthawing the platelets enriched fractions, by S/D treatment, bysonication [Slezak et al., (1987) J. Exp. Med. V 166 p 489-505], byFrench press [Salganicoff et al., (1975) Biochem. Biophys. Acta v385 p394-411] and/or by any other method known in the art.

In one embodiment of the invention, lysis of the platelets is carriedout by freezing and thawing the platelets-enriched fractions followed bycarrying out an S/D treatment. Typically, lysis of the plateletsproduces a cell free platelet lysate.

The term “viral-safe biological liquid mixture” refers to a mixtureand/or composition which was subjected to at least two orthogonal viralinactivation treatments.

The term “viral-safe platelet extract” refers to an extract which wassubjected to at least two orthogonal viral inactivation treatments.

The term “viral inactivation treatment” and “inactivating viruses”refers to a situation wherein viruses are maintained in the solution butare rendered non-viable e.g. by dissolving their lipid coat; and/or tothe situation wherein viruses are physically removed from the solutione.g. by size exclusion techniques.

The term “orthogonal viral inactivation treatment” involves carrying outat least two different and independent treatments for inactivatingviruses. A combination of two or more of the following non limitingtreatment examples can be used: heat inactivation, Solvent/Detergent(S/D), nanofiltration, Low pH treatment, UV irradiation and Sodiumthiocyanate treatment.

“Solvent detergent (S/D) viral inactivation treatment” typically refersto a process that inactivates enveloped or lipid-coated viruses bydestroying their lipid envelope. The treatment can be carried out by theaddition of detergents (such as Triton X-45, Triton X-100 or polysorbate80) and solvents [such as tri(n-butyl) phosphate (TnBP), di- ortrialkylphosphates]. The solvent-detergent combination used todeactivate lipid coated viruses may be any solvent-detergent combinationknown in the art such as TnBP and Triton X-100; polysorbate 80 andSodium cholate and other combinations.

The concentration of the solvent(s) detergent(s) used can be thosecommonly used in the art, for example as carried out in U.S. Pat. No.5,094,960A, U.S. Pat. No. 4,789,545A. In one embodiment of theinvention, a combination of >0.1% TnBP and >0.1% Triton X-100 is used.In another embodiment of the invention, a combination of 1% Triton X-100and 0.3% TnBP is used. Typically, the conditions under which thesolvent-detergent inactivates the viruses consist of 10-100 mg/ml ofsolvent detergent at a pH level ranging from 5-8, and a temperatureranging from 2-37° C. for 30 minutes to 24 hours. However, other solventdetergent combinations and suitable conditions will be apparent to anyperson versed in the art. This inactivation includes treatment with S/Dand removal of the S/D.

“Heat inactivation” typically refers to a process by which heat destroysboth lipid-enveloped and non-enveloped viruses. “Heat inactivation” isinterchangeable with the term “Pasteurization”. The heat inactivationcan be carried out at a temperature in the range of 59.5 to 60.5° C. fora period of 9 to 10.5 hours e.g. the inactivation can be carried out at60° C. for 10 hours. Stabilizers such as sucrose and glycine can beadded into the material during the heat inactivation step.

“Nanofiltration” typically refers to a process by which lipid-envelopedand non-enveloped viruses are excluded from the sample by usingnanometer-scale filters such as Planova™ 20N, 35N and 75N;Viresolve/70™, Viresolve/180™. The filters can have a pore size of lessthan 70 nm, preferably between 15 and 50 nm. However, any membranehaving a pore size sufficient to reduce or eliminate viruses from thesample can be employed in nanofiltration. Viruses removed bynanofiltration can be enveloped [e.g. HIV, hepatitis B virus, hepatitisC virus, West Nile Virus, cytomegalovirus (CMV), Epstein-Barr virus(EBV), herpes simplex virus], and non enveloped (e.g. hepatitis A virus,paravirus B 19, Polio virus).

Low pH treatment is typically effective against enveloped viruses. Inone embodiment of the invention, the platelet lysate is subjected to alow pH, typically to a pH of 4, and lasts anywhere between 6 hours and21 days. “Low pH treatment” is interchangeable with the term “acidic pHinactivation”.

In one embodiment of the invention, the first viral inactivation step ofthe extract preparation comprises solvent-detergent (S/D) treatment ofthe platelets for eliminating enveloped viruses. The S/D treatment alsopromotes lysis of the platelets and release of their content into thesolution. For optimal envelope viral inactivation, a sub-step includingaggregates removal (e.g. by filtration) can be carried out during theS/D treatment step.

The term “platelet-enriched fraction from more than one donor” refers toa platelet-enriched material which is obtained from at least twoindividuals. The individuals can be human or other mammalians. In someembodiments, platelets are collected from 5 to 12 donors.

The term “platelet-enriched fraction” refers to a plasma compositionhaving a concentration of platelets above that of the concentration ofplatelets normally found in blood. In a particular embodiment, plateletconcentration is above the normal baseline concentration of platelets,for example, about 200.000 platelets/μL. For example, the plateletconcentration may be at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, or 100 times or more the normalconcentration in blood. In certain embodiments, the platelet-enrichedfraction has a platelet concentration of greater than about 200,000platelets/μL, 300,000 platelets/μL, 400,000 platelets/μL, 500,000platelets/μL, 600,000 platelets/μL, 700,000 platelets/μL, 800,000platelets/μL, 900,000 platelets/μL, 1,000,000 platelets/μL, 1,100,000platelets/μL, 1,200,000 platelets/μL, 1,300,000 platelets/μL, 1,400,000platelets/μL, 1,500,000 platelets/μL, 1,600,000 platelets/μL, 1,700,000platelets/μL, 1,800.000 platelets/μL, 1,900,000 platelets/μL, or2,000,000 platelets/μL.

Fractions from which the platelet-enriched material can be obtained frominclude, but are not limited to, blood fractions, plasma fractions,washed and leukocyte-reduced platelets from aphaeresis, and plateletsfrom aphaeresis. In one embodiment, washed and/or leukocyte-reducedplatelets pooled from multiple donors is used as the starting materialfor preparation of the platelet extract.

Using washed platelets as the starting material for preparing theextract enables obtaining a non-clottable platelet extract with reducedplasma impurities (e.g. reduced IgG and fibrinogen levels).

Typically, the term “platelet starting material” relates toplatelet-enriched fractions obtained from more than one donor for use inthe method of the invention. The platelet-enriched fractions can be, forexample, separated from units of whole blood, from blood fractionsand/or from plasma fractions. The platelet-enriched fractions can beobtained from aphaeresis donations. The starting material can be washedand/or leukocyte-reduced. In one embodiment of the invention, theplatelet-enriched fractions are washed and leukocyte-reduced and areobtained from aphaeresis donations. In one embodiment, the minimalnumber of platelets in an aphaeresis leukocyte-reduced collected unit isabout or more than 3.0×10¹¹ as specified in the “Circular of Informationfor the Use of Human Blood and Blood Components”.

The term “washed platelets” refers to platelets which were subjected toa washing step. During the washing procedure there can also be losses ofplatelets. The washing can be carried out using 0.9% sodium chloridewith or without small amounts of dextrose. The washing procedure can becarried out as elaborated in the “Circular of Information for the Use ofHuman Blood and Blood Components”. In one embodiment of the invention,the washing is carried out as follows: a platelet material unit iscentrifuged under gentle conditions. Then, the supernatant is discardedand the platelet pellet is washed at least twice (with centrifugationbetween the washes) with saline under gentle conditions. The washed andre-suspended platelets can be frozen until used in the method of theinvention.

The term “leukocyte-reduced” refers to a content of leukocyte which islower than the content of leukocyte in whole blood (content in wholeblood is about 1 to 10×10⁹ white cells per blood unit). Any leukocytesreduction methods, e.g. by filtration, can be used to obtain aleukocyte-reduced unit. The reduction in leukocytes can be carried outduring aphaeresis. Typically, a leukocyte-reduced unit of plateletswhich contains less than about 5×10⁶ leukocytes is used as the startingmaterial for the preparation of the platelet extract.

The term “aphaeresis” typically refers to the withdrawal of blood from asingle donor, with a portion (e.g. platelets) being separated andretained and the remainder retransfused into the donor. One unit ofaphaeresis platelets obtained from a single donor can contain about orhigher than 3.0×10¹¹ platelets. In one embodiment of the invention, oneunit of aphaeresis platelets obtained from a single donor contains up to6.0×10¹¹ platelets. Oftentimes, when there are more than 6×10¹¹platelets in one donation, the donation unit is split into two separatebags.

The term “amphiphilic polymer” or “amphipathic polymer” is a polymerpossessing both hydrophilic (having an affinity for water, polar) andlipophilic (having an affinity for lipids) properties. The lipophilicgroup is typically a large hydrocarbon moiety, such as a long chain ofthe form CH3(CH2)n, with n>4. In one embodiment, the hydrophilic groupfalls into one of the following categories:

1. Charged groups:

Anionic. Examples, with the lipophilic part of the molecule representedby an R, are:

carboxylates: RCO2-;

sulfates: RSO4-;

sulfonates: RSO3-.

phosphates: The charged functionality in phospholipids.

Cationic. Examples:

amines: RNH3+.

2. Polar, uncharged groups. Examples are alcohols with large R groups,such as diacyl glycerol (DAG), and oligoethyleneglycols with long alkylchains.

Often, amphiphilic species have several lipophilic parts, severalhydrophilic parts, or several of both. Proteins and some blockcopolymers are such examples.

Amphiphilic compounds have lipophilic (typically hydrocarbon) structuresand hydrophilic polar functional groups (either ionic or uncharged).

As a result of having both lipophilic and hydrophilic portions, someamphiphilic compounds may dissolve in water and to some extent innon-polar organic solvents. When placed in an immiscible biphasic systemconsisting of aqueous and organic solvent the amphiphilic compound willpartition in the two phases. The extent of the hydrophobic andhydrophilic portions determines the extent of partitioning.

Non limiting examples of non toxic amphiphilic polymers are Polyethyleneglycol (PEG), polyethylene oxides (PEO),Poly(2-acrylamidohexadecylsulfonic acid (PAMC16S),lipopoly(2-methyl-2-oxazoline)s (LipoPOxs), Hydroxyethyl starch (HES),amphiphilic polymers derived from Tris(hydroxymethyl)-acrylamidomethane(THAM) Cationic polymers used for gene therapy like Poly-L-Lysin (PLL)-and Polyethyleneimine (PEI)-based polymers.

Typically the term “non toxic” refers to a product, substance, orchemical compound that is non-toxic to a patient at the dosages andconcentrations employed, and will not cause adverse health effects,either immediately or over the long-term. A non-toxic or physiologicallysafe compound is understood as a compound with an LD50 (rat) of ≧500mg/kg, better ≧950 mg/kg and best ≧2000 mg/kg.

In one embodiment of the invention, the amphiphilic polymer is ahydrocarbon based surfactant.

The term “hydrocarbon based surfactant” is hydrocarbon compound thatlowers the surface tension of a liquid, the interfacial tension betweentwo liquids, or that between a liquid and a solid. Hydrocarbonsurfactants may act as detergents, wetting agents, emulsifiers, foamingagents, and dispersants.

The term “contacting” is used herein in its broadest sense and refers toany type of combining action which e.g. brings the amphiphilic polymerinto sufficiently close proximity with the factors of interest present(e.g. growth factors, cytokines, chemokines and/or trophic factors) inthe S/D treated material or source such that a binding interaction willoccur between the amphiphilic polymer and the factors. Contactingincludes, but is not limited to, mixing, admixing and/or adding theamphiphilic polymer into the S/D treated material and/or adding theamphiphilic polymer into the buffer used to wash the HIC column, and/orin the oil used to extract the S/D.

The polymer can have an average molecular weight of from 200 to below50000 Daltons. In one embodiment of the invention, the amphiphilicpolymer is polyvinylpyrrolidone (PVP). PVP can be in a range of 12-30K,or about 12, 13, 14, 15, 16, 117, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30K.

In one embodiment of the invention, the amphiphilic polymer ispolyvinylpyrrolidone having an average molecular weight in the range of3500 to 40000 Dalton. E.g. the PVP used can have an average molecularweight of 3500 Dalton and/or a K-Value in the range of 10.2-13.8; anaverage molecular weight of 8000 Dalton and/or a K-Value in the range of16.0-18.0; an average molecular weight of 30000 Dalton and/or a K-Valuein the range of 22.5-27.0; or an average molecular weight of 40000Dalton and/or a K-Value in the range of 27.0-32.4. A combination ofdifferent amphiphilic polymers and/or the same polymer having adifferent average molecular weight can be used to contact the S/Dtreated material. In one embodiment, PVP having an average molecularweight of 30000 Daltons is added into the S/D treated material prior S/Dremoval e.g. prior to loading the material onto the column; and then PVPhaving a molecular weight of 30000 Daltons is added into the buffer usedto wash the column.

It was found that PVP K25 concentration of (0.3 mM) 1% or higherresulted in the presence of S/D material, namely Triton X-100, in thepost-SDR material above the acceptable limit.

In one embodiment of the invention, the amphiphilic polymer is contactedwith the S/D treated material within a concentration range of 0.01%(w/w) to lower than 1% (w/w); in the range of 0.1% (w/w) to lower than1% (w/w); or in the range of 0.1% (w/w) to 0.5% (w/w).

In a next step, an S/D removal step is carried out. The term“solvent-detergent removal (S/D removal)” refers to the removal of thebulk of the solvent-detergent used in the S/D treatment. The removal ofsolvent-detergent comprises using hydrophobic interaction chromatographycolumn (HIC) e.g. C-18 silica packing material and SDR(Solvent-Detergent removal) HyperD; oil extraction; a combinationthereof or any other method known in the art.

In one embodiment of the invention, oil extraction is used to remove thesolvent-detergent.

Liquid-liquid extraction, also known as “solvent extraction” and“partitioning”, or “depletion partition” is a method to separatecompounds based on their relative solubilities in two differentimmiscible liquids. It is an extraction of a substance from one liquidphase into another liquid phase. Two immiscible liquids can be oil andan aqueous liquid. Oftentimes in this case removal of a substance usingoil and aqueous partition is referred as “oil extraction”. Addition ofoil to an aqueous solution comprising solvent detergent, mixing andallowing partition between water and oil will lead to leave a major partof the solvent detergent in the oil phase.

In another embodiment of the invention. SDR HyperD, which is achromatographic packing made of silica beads in which the pore volume isfilled with a three-dimensional cross-linked hydrophobic acrylicpolymer, is used to remove the solvent-detergent. The SDR HyperDadvantageously involves a mixed-mode adsorption of hydrophobicinteraction and is associated with a molecular exclusion effect[Guerrier L et al. “Specific sorbent to remove solvent-detergentmixtures from virus-inactivated biological fluids”. J Chromatogr BBiomed Appl. 1995 Feb. 3; 664(1):119-125].

The term “hydrophobic interaction chromatography (HIC)” refers e.g. to acolumn packed with a hydrophobic polymer resin. Generally the mixture isallowed to travel through the column comprising the packed resin at acertain flow rate, and the S/D material is being removed. HIC can becarried out batch-wise.

Hydrophobic resins are well known in the art. Non limiting examples aree.g. C-18 silica packing material and SDR (Solvent-Detergent removal)HyperD.

The hydrophobic interaction chromatography can be carried out by amethod comprising the following steps: loading the S/D-treated andpolymer-contacted material to HIC; washing with an aqueous solutionoptionally comprising a low concentration of organic solvent (e.g.ethanol at a concentration range of 5-15%) and/or a salt (e.g. NaCl at aconcentration of 0.2-1.2M); and collecting the wash material.

Non limiting examples of salts are KCl, MgCl₂, CaCl₂ and the like.

Non limiting examples of organic solvents are isopropanol, glycerol,ethylene glycol and the like.

The term “loading to HIC” refers to applying the material to the column.However, if desired, the same resin can be used “batch-wise” to removethe S/D material. As used herein, “batch-wise” generally refer to atechnique in which the resin and the mixture are incubated together e.g.in a stirred tank, batch reactor or a vessel, and the adsorption iscarried out in a continuous manner. In one embodiment of the invention,the mixture is contacted with the resin in a vessel e.g. a tube, andafter an incubation period, the vessel is centrifuged and thesupernatant comprising the platelet-derived factors is collected (theS/D material is present within the precipitate). The batch method can becarried out in a vessel or a batch reactor.

The term “S/D-treated and polymer-contacted material” means a substancethat was subjected to an S/D for viral inactivation and contacted withan amphiphilic polymer as defined above.

The material loaded to the HIC column can be dissolved in a bindingbuffer. The column can be equilibrated prior to loading the materiale.g. by washing the column with the binding buffer.

The term “equilibrate” refers to allowing and/or adjusting the column toreach a specific buffer condition such as a specific pH level, specificamphiphilic polymer concentration and ionic strength. In one embodimentof the invention, the adjustment of the column is carried out by washingthe column with an equilibration buffer having a predetermined pH leveland ionic strength prior to loading the S/D-treated andpolymer-contacted material onto the column. In one embodiment of theinvention, the equilibration buffer comprises 20 mM sodium acetate and10 mM glycine at pH 6.8-7.4; 0.2% (w/w from the total volume) humanserum albumin (HSA) and 0.1% amphiphilic polymer.

In one embodiment of the invention, a method for removingsolvent-detergent (S/D) from a biological liquid mixture comprises thesteps of: contacting the S/D treated mixture with a non toxicamphiphilic polymer; removing the S/D from the mixture by subjecting themixture to hydrophobic interaction chromatography (HIC) and/or oilextraction and collecting a material comprising a flow through fractionfrom HIC and/or liquid fraction from oil extraction.

In a further embodiment, the HIC comprises the steps of: washing with asolution comprising organic solvent and/or a salt.

In a further embodiment of the invention, the collected materialincludes the flow through fraction combined with the wash fraction ofHIC.

The term “binding buffer” refers to the buffer used during loading ofthe S/D-treated and polymer-contacted material onto the chromatographycolumn. Oftentimes, the equilibration buffer used to adjust the columnprior and/or during loading of the material is termed binding buffer. Inone embodiment of the invention, the binding buffer comprises 20 mMsodium acetate and 10 mM glycine at pH 6.8-7.4; 0.2% (w/w from the totalvolume) human serum albumin (HSA) and 0.1% amphiphilic polymer. HIC canalso comprise the steps of: washing HIC with the equilibration bufferand/or the binding buffer, and collecting an unbound material.

Flow through or unbound material typically refers to the fractioncollected following washing of the loaded column with the same bufferused for equilibration and/or the buffer used for loading the mixtureonto the column (“binding buffer”).

The term “washing” refers to washing the column during an S/D removalstep with a solution or condition equal or different from the solutionor condition used to load and/or equilibrate the column, and/or equal ordifferent from the solution used in a previous step. The washingconditions are such that S/D substantially remains bound to thecolumn/resin whereas the factors are washed/unbound.

Washing conditions, may involve an increase in salt concentration and/orincluding an organic solvent within the solution.

The platelet extract may comprise a mixture of growth factors, trophicfactors, chemokines and/or cytokines.

The term “growth factor” typically refers to an agent that promotescellular growth, proliferation and/or differentiation. Examples ofgrowth factors include, but are not limited to, transforming growthfactor (TGF) e.g. TGF-b1, fibroblast growth factor (FGF) e.g. bFGF,vascular endothelial growth factors (VEGF), platelet-derived growthfactor (PDGF) e.g. PDGF-AB, and the like.

The term “trophic factors” typically refers to an agent that stimulatesdifferentiation and/or survival of cells. Examples of trophic factorinclude, but are not limited to, adhesion molecules, bone morphogeneticproteins, cytokines, eph receptor tyrosine kinase, epidermal growthfactors, fibroblast growth factors (FGF), GDNF, heparin-binding growthfactors, insulin-like growth factors, neurotrophins, semaphorins,transforming growth factors (TGF) β, tyrosine kinase receptor ligands,and the like.

The term “cytokines” typically refers to cell derived signaling proteinmolecules that are secreted by cells and are a category of signalingmolecules used extensively in intercellular communication. Immune cellsrelease cytokines.

A platelet factor may have a growth activity, a cytokine, a chemokineactivity and/or a trophic activity.

In another aspect, the invention relates to an active and viral-safe (atleast double viral inactivated) platelet extract derived from multipledonors obtainable according to the methods of the invention; and to itsuse. The viral-safe platelet extract comprises a mixture of biologicallyactive platelet cell growth factors, chemokines, cytokines and/ortrophic factors.

In another aspect, the invention relates to a method for removing anamphiphilic toxic molecule such as solvent-detergent (S/D) from abiological liquid mixture comprising the amphiphilic toxic molecule. Themethod comprises the steps of providing the biological liquid mixturecomprising the amphiphilic toxic molecule; contacting the mixture with anon toxic amphiphilic polymer such as PVP; and removing the amphiphilictoxic molecule from the mixture.

An amphiphilic toxic molecule typically includes, but is not limited to,Triton X-45, polysorbate (e.g. polysorbate 20, polysorbate 80). Brij(polyethylene glycol lauryl ether, e.g. Brij 30, Brij 35, Brij 58),IGEPAL (octylphenoxypolyethoxyethanol. e.g. IGEPAL CA-630) and the like.

The term “biological liquid mixture” refers to any type of liquidsubstance obtained from a biological source and/or a liquid thatcomprises recombinant ingredients and/or recombinant platelet derivedfactors e.g. chemokines, growth factors, cytokines trophic factors or acombination thereof. “A biological source” typically includes, but isnot limited to, preparations obtained from body fluids such as wholeblood plasma or blood fractions e.g. cryodepleted plasma,cryoprecipitate, plasma or serum; semen; sputum; feces; sweat; saliva;nasal mucus; cerebrospinal fluid; a platelet derived fraction such asPlatelet Rich Plasma releasate PRP-R (PRP-releasate); and urine, as wellas liquids obtained from cell cultures, containing biological substancessecreted by the cells into the preparation, or containing substanceswhich originally were present inside the cells, and were released to theliquid preparation due to various manipulations such as lysing of thecells or activating of the cells.

The term “cryoprecipitate” refers to a blood component which is obtainedfrom frozen plasma prepared from whole blood. A cryoprecipitate can beobtained when frozen plasma is thawed in the cold, typically at atemperature of 0-4° C., resulting in the formation of precipitatedsupernatant that contains fibrinogen and factor XIII. The precipitatecan be collected, for example by centrifugation. The solution of BACcomprises further Factor VIII, fibronectin, von Willebrand factor (vWF),vitronectin, etc. for example as described in U.S. Pat. No. 6,121,232and WO9833533.

In one embodiment, the method for removing S/D according to theinvention can be used after a process for viral inactivation of abiological liquid preparation. Biologically derived liquid preparationssuch as blood and plasma preparations are used as raw materials fromwhich a plurality of biologically useful compounds can be purified.Examples of such compounds include immunoglobulin, factor VIII, albumin,a 1 anti trypsine, Factor IX, factor XI, PPSB, fibrinogen, and thrombin(prothrombin). In addition, various biological products such ashormones, growth factors, enzymes, ligands and antibodies are isolatedfrom biological preparations obtained from cell cultures.

Yet, in another aspect, the invention relates to a pharmaceuticalcomposition comprising PVP at a concentration range of 0.07 to 6 mM anda platelet derived protein composition comprising chemokines, growthfactors, cytokines, trophic factors or a mixture thereof.

It is shown here that performing removal of S/D using PVP inconcentration of 0.9 mM (6 mM in the final product) resulted in removalof 98% of the triton to a final concentration of 170 ppm. The traces ofTriton X-100 that remained in the material after the column were removedin the downstream process until no traces of triton were detected in thefinal product. Considering these results using PVP in concentrationhigher than 0.9 mM may result in elution of triton from the column atconcentration that cannot be removed downstream.

The term “pharmaceutical composition” refers to any compound orcomposition of matter or combination of constituents, which whenadministered to a subject induces a physiologic and/or biological effect(e.g. induction of cell proliferation, cell motility, cell-cellinteractions, and/or cellular morphological changes) by local and/orsystemic action.

It was found that using PVP K12 and PVP K25 during S/D removal it ispossible to obtain a composition which has ratios of the factors whichare comparable to the ratios in the WAP starting material.

It is disclosed a composition having PDGF-AB/TGF-β1 in the range ofabout 0.3-0.4; PDGF-AB/VEGF in the range of about 41 to about 102;TGF-β1/bFGF in the range of about 1500 to about 1700; and/or VEGF/bFGFin the range of about 6.0 to 12.5.

The term “subject”, as used herein, includes animals of mammalianorigin, including humans. In one embodiment, the subject is a human.

The viral-safe platelet extract prepared according to the invention canbe used for any therapeutic purpose.

The extract of the invention is suitable for any therapeutic use e.g.for promoting healing of injured tissue in a subject. The plateletextract can be used as is for injection into a target area or forintravenous administration; applied onto/administered into bandages,foams, pads and matrices and/or can be used in combination with fibrinsealant for topical applications. The extract can be released into/ontoa desired location from different delivery agents such as bandages,pads, foams and matrices. The agents can be made of natural and/orsynthetic materials. Examples of such materials include, but are notlimited to, polymers, hydrogels, Polyvinyl alcohol (PVA), polyethyleneglycol (PEG), hyaluronic acid, chondroitin sulfate, gelatin, alginate,collagen matrices, carboxymethylcellulose, dextran,poly(2-hydroxyethylmethacrylate) [PHEMA], agar, oxidized regeneratedcellulose (ORC), self assembled peptides [SAPs], poly(glycolic) acid,poly(lactic) acid, fibrin and combinations thereof.

It was found that administering fibrin sealant in combination with aplatelet extract obtained according to the invention which comprises PVPhad a significant positive effect on healing in-vivo: it promoted skinflap adherence, and accelerated the healing process, as shown usinghistology, compared to fibrin sealant alone or the sham group (animalsthat underwent the same flap creation procedure, but did not have anytreatment applied prior to flap closure).

The term “any therapeutic purpose” refers to any curative or preventivetreatment; for cosmetic use; and/or for any disease, disorder orcondition in a subject. Exemplary therapeutic purposes include, but arenot limited to, to improve graft integration; accelerating internal orexternal wound healing. i.e., causing the wound to heal rapidly ascompared to an untreated wound or to other known wound treatments;treating any injury or condition that requires stimulating angiogenesis,mitogenesis, cell proliferation, neutrophils and macrophages, collagensynthesis, migration, wound contraction, extracellular matrix synthesis,epithelialization and chemotaxis; injury or condition that requirestissue generation, regeneration or reorganization, epithelialization,formation of new blood vessels, or angiogenesis; for decreasing scarformation; reducing post operative complications and morbidity; forhealing soft tissue e.g. skin wounds e.g. for healing surgical skin flapfailure, cuts or ulcers. The composition disclosed can be administeredby topical or parenteral route.

The platelet extract can be used in various surgical fields such as, butnot limited to, orthopedic surgery (e.g. bone repair, articularcartilage repair, knee arthroplasty, lumbar spinal fusion, and inintervertebral disc degeneration); dental surgery; dentistry andmaxillofacial surgery (e.g. consolidation of titanium implants,maxillary sinus augmentation and bone remodeling); for muscle, tendonand ligament repair; facial plastic and reconstructive surgery; chronicskin wound healing, skin burn healing, ophthalmology; facial nerveregeneration, peripheral nerve repair, central nervous system (CNS)repair (spine and/or brain surgery), optic nerve repair, nervecompression syndrome repair, cranial nerve repair, sciatic nerve repair;cardiac; gastrointestinal surgery and bariatric surgery. The extract canbe administered onto a surface of a body part of a patient. The term“surface” refers to an external surface that can be seen by unaidedvision and to a surface of an internal body part which is a part of theinternal anatomy of an organism. External surfaces include, but are notlimited to, the skin of the face, throat, scalp, chest, back, ears,neck, hand, elbow, hip, knee, and other skin sites. Examples of internalbody parts include, but are not limited to, body cavity or anatomicalopening that are exposed to the external environment and internal organssuch as the nostrils; the lips; the ears; the genital area, includingthe uterus, vagina and ovaries; the lungs; the anus; the spleen; theliver; the cardiac muscle, and the gastrointestinal tract. The surfacecan be a bleeding or a non-bleeding site. Alternatively, the extract canbe administered by injection e.g. intradermally, intraperitonealy,subcutaneously, intrathecally, intrasternally, intracranially,intramuscularly, and/or intravenously. The extract can also beadministered by infusion.

The invention also provides a method of treating inflammation; tissuehealing; organ reconstruction and/or tissue regeneration comprisingadministering to a subject in need a therapeutically effective amount ofan extract according to the invention.

The extract according to the invention can also be used for facilitatinggrowth, proliferation, differentiation and/or maintenance of variouscell types e.g. stem cells. For this purpose, the extract can be usedalone or in combination with fibrin sealant in in vivo and/or in vitroapplications. In one embodiment, the extract can be used together with abiocompatible implant e.g. for tissue engineering in vivo, as well asfor in vitro cell culturing.

The term “a therapeutically effective amount” refers to the doserequired to prevent or treat (relieve a symptom or all of the symptoms)a disease, disorder or condition. The effective amount can be measuredbased on any change in the course of the disease in response to theadministration of the composition. The effective dose can be changeddepending on the age and weight of the subject, the disease and itsseverity (e.g. early or advanced stage) and other factors which can berecognized by the skilled in the art. The extract can also comprise apharmaceutically acceptable excipient. As used herein the term“excipient” refers to an inert substance which is added into theextract. Typically, an excipient is a material used in the finalformulation of a pharmaceutical composition. The excipients can beadded, for example, in order to ensure that the active substances retaintheir chemical stability and/or biological activity upon storage, to aidthe manufacturing process and/or for aesthetic reasons e.g. color. Theadded excipient is generally safe and non-toxic.

The platelet extract according to the invention can be used incombination with a surgical sealant. Different types of surgicalsealants can be used in combination with the platelet extract,including, but not limited to, a biological sealant (such as a fibrinsealant prepared with fibrinogen and thrombin components); a syntheticsealant such as acrylates, cyanoacrylates, and polyethylene glycol (PEG)polymers; and a semisynthetic sealant e.g. made from a combination ofbiological and synthetic materials such asgelatin-formaldehyde-resorcinol (GFR) glue. In one embodiment of theinvention, the platelet extract is used in combination with fibrinsealant components. In another embodiment of the invention, the plateletextract is used with a synthetic sealant.

If desired, the platelet extract obtained by the method of the inventioncan be dried e.g. by lyophilization, supercritical fluid technology,spray freeze drying, spray coating, modifications of spray coating suchas drying with conventional spouted bed, and other drying methods basedon solvent evaporation without atomization (such as vacuum drying,Xerovac1, foam drying, film drying) or spray drying. Prior to drying,the extract can be formulated with a cryoprotectant.

The term “cryoprotectant” refers to a substance which is added tosolutions in order to retain the chemical stability and/or biologicalactivity of the active components (e.g. growth factors, chemokines,cytokine and/or trophic factors) during freezing. Non limiting examplesof cryoprotectant include, but are not limited to, carbohydrates such asMonosaccharides: include glucose (dextrose), fructose (levulose),galactose, and ribosedisaccharides Disaccharides: sucrose, lactose,maltose and trehalose and Disaccharides oligosaccharides another groupare the poliols Sugar alcohols: Maltitol, Mannitol, sorbitol, xylitoland isomalt. Apart of carbohydrates other polymers such as Polyethyleneglycol (PEG) can also be used as cryoprotectants such as polyethyleneoxide (PEO) or polyoxyethylene (POE), or amino acids and polyamines.

The term “lyophilization” typically refers to the process of freezing asubstance and then reducing the concentration of water e.g. bysublimation to levels which do not support biological or chemicalreactions. The resulting lyophilized biological material may be storedfor a relatively long period of time. Following storage, the lyophilizedmaterial can be used as a powder or can be reconstituted by the additionof various volumes of an aqueous solution. The volume added duringreconstitution can be similar to the volume of the solution beforelyophilization, lower (resulting in a concentration of the extractcompared to the volume of the starting material) or higher (resulting ina dilution of the extract compared to the volume of the startingmaterial). If desired, the platelet extract can be kept frozen or assolid e.g. lyophilized for prolonged storage or for use as a powder.

For example, the platelet extract obtained by the method of theinvention can be kept frozen e.g. at −18° C. or at lower temperature, oras solid (e.g. lyophilized) for prolonged storage. The platelet extractcan also be refrigerated e.g. at a temperature of 2° C. to 8° C.

The lyophilized extract can be used as solid or can be reconstituted ina pharmaceutically acceptable carrier prior to use. The term a“pharmaceutically acceptable carrier” refers to any diluent and/or avehicle which is suitable for human administration or for animaladministration. The carrier can be selected from any of the carriersknown in the art such as, but not limited to, saline, sodium chloridesolution, lactated ringers (LR), 5% dextrose in normal saline, and waterfor injection. If administered with fibrin sealant, the extract can bereconstituted in one of the sealant components (thrombin or fibrinogen)or can be reconstituted separately in another diluent or vehicle.

Of advantage, the lyophilization cycle and the formulation can allow fora very fast reconstitution of the extract e.g. within fibrin sealante.g. to facilitate hemostasis and healing which calls for an emergencyuse, thus in this case the reconstitution is beneficially done withinseconds. In one embodiment of the invention, albumin is used in theformulation to allow fast reconstitution.

The extract obtained according to the method of the invention can beconcentrated. The concentration can be carried out at any step e.g.immediately after the S/D removal step or at a later step. Concentrationcan be achieved by diafiltration of the material and/or reconstitutionof a lyophilized extract in a lower volume compared to the volume of theextract prior to its lyophilization.

The invention provides a kit. The kit may comprise a recipientcomprising the extract according to the invention. The extract can be ina solid form e.g. lyophilized, as a solution or in frozen form. In thecase that the extract is provided in solid form, the kit can furthercomprise a recipient with a pharmaceutically acceptable carrier forreconstituting the solid extract. The kit may further comprise one ormore syringes and/or syringe needles for injecting the extract to thepatient. The kit can comprise instructions for use. The instructions maydescribe how to administer the extract to a patient. The invention alsorelates to a kit comprising recipients containing the components of thefibrin sealant, the synthetic sealant, and/or another possible deliveryagent; a recipient containing the extract of the invention; andinstructions for use. Optionally, the extract of the invention can be inthe recipient of one component of the fibrin sealant. Also, theinvention relates to a kit comprising a recipient containing thelyophilized extract, a recipient containing a reconstitution solution orcarrier and instructions for use.

The fibrin sealant components can be prepared from blood compositions.The blood composition can be whole blood or blood fractions, i.e. aproduct of whole blood such as plasma.

In one embodiment of the invention, the fibrinogen component iscomprised from a biologically active component (BAC) which is a solutionof proteins derived from blood plasma which can further comprisetranexamic acid and arginine or lysine or mixtures or arginine andlysine, or their pharmaceutically acceptable salts. BAC can be derivedfrom cryoprecipitate, in particular concentrated cryoprecipitate.

The composition of BAC can comprise stabilizers such as argininehydrochloride. Typically, the amount of fibrinogen in BAC is in therange of from about 40 to about 60 mg/ml. The amount of tranexamic acidin the solution of BAC can be from about 80 to about 110 mg/ml. Theamount of arginine hydrochloride can be from about 15 to about 25 mg/ml.

Optionally, the solution is buffered to a physiological compatible pHvalue. The buffer can be composed of glycine, sodium citrate, sodiumchloride, calcium chloride and water for injection as a vehicle. Glycinecan be present in the composition in the amount of from about 6 to about10 mg/ml, the sodium citrate can be in the range of from about 1 toabout 5 mg/ml, sodium chloride can be in the range of from about 5 toabout 9 mg/ml and calcium chloride can be in the concentration of about0.1-0.2 mg/ml. Optionally, the BAC component can be diluted to comprise3-60 mg/ml fibrinogen. The dilution can be carried out e.g. using asolution comprising glycine, sodium citrate, sodium chloride, calciumchloride and water for injection.

In one embodiment, the thrombin component used can be in a range of100-1200 IU/ml.

During application of the liquid fibrin sealant formulation onto thedesired location, the fibrinogen containing component and the thrombincontaining component may be applied in any desired range of ratios. Forexample, when the concentration of the fibrinogen component is 3-60mg/ml and the thrombin concentration is about 10-1200 IU/ml the twocomponents can be mixed in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,respectively, and so on. In one embodiment of the invention, thecomponents of the liquid fibrin sealant are applied in a ratio of 3:1.

In another embodiment, the concentration of plasminogen and plasmin inthe BAC composition is lowered to equal or less than 15 μg/ml like forexample 5 μg/ml or less plasminogen e.g. using a method as described inU.S. Pat. No. 7,125,569 and WO02095019. In this case addition oftranexamic acid, aprotinin or any other fibrinolytic inhibitors into theBAC is not needed.

It is also possible that the fibrin sealant comprises components whichencourage the formation of the clot, such as Ca²⁺, Factor VIII, FactorXIII, fibronectin, vitronectin, von Willebrand factor (vWF) which can beprovided as a separate component or formulated with the fibrin sealantcomponents.

Fibrin sealant components derived from blood compositions are typicallypurified from infective particles. The purification procedure can becarried out by nanofiltration; solvent/detergent treatment and/or by anyother method known in the art.

The term “infective particle” refers to a microscopic particle, such asmicro-organism or a prion, which can infect or propagate in cells of abiological organism. The infective particles can be viral particles.

The platelet extract prepared according to the invention can be used incombination with various cell types e.g. fibroblast and stem cells e.g.endothelial stem cells e.g. HUVEC. The cell type can be determinedaccording to the intended therapeutic use. For example, for regenerationof intervertebral disc, a cell composition comprisingnotochordal-derived cells can be used. For induction of angiogenesis,endothelial stem cells can be used.

The following examples are illustrative but not limiting.

EXAMPLES

Materials and Methods

Washed aphaeresis platelets leukocyte-reduced (WAP) preparation.

Platelet material units (platelets aphaeresis leukocyte-reduced units)were collected and processed according to the “Circular of Informationfor the Use of Human Blood and Blood Components” (December 2009) andconformed to applicable federal statuses and regulations of the FDA andUS Department of Health. Each unit had a volume of approximately 200 ml.Each unit was drawn from a single donor who was screened and foundacceptable for donation of a transfusable blood component based on FDAregulations, requirements and guidelines. Included were only units whichwere found non-reactive for red blood cell antibodies and negative forthe following viruses by using FDA-approved kits and methods: HepatitisB virus surface antigen; Antibody to hepatitis B virus core antigen;Hepatitis C virus antibody; Human T-cell lymphotrophic virus type 1 and2 antibody; Human immunodeficiency virus types 1 and 2 antibody; HIV-1by nucleic acid technology testing (NAT); HCV RNA by NAT; West NileVirus RNA by NAT; and Serological test for syphilis. The minimal numberof platelets in an aphaeresis leukocyte-reduced collected unit was asspecified in the Circular of Information: ≧3.0×10¹¹ (the number ofplatelets in a single whole blood unit is ≧5.5×10¹⁰).

All units were maintained under the recommended conditions fortransfusion (see the direction for use for the blood collection,processing, and storage system approved by the FDA) until the washingstep.

Washing Procedure.

Each unit was washed under aseptic conditions as follows:

-   -   1. Each unit was centrifuged at 4658×g for 6 minutes (rotor        break was not used) at room temperature. Under these gentle        conditions breakage of the cells was avoided.    -   2. The supernatant was discarded and the platelet pellet was        re-constituted in 200 ml sterile docked saline (a way of        transferring liquids between containers in a closed system to        maintain aseptic conditions).    -   3. A second centrifugation was carried out in the same        conditions specified in step 1.    -   4. The saline was discarded and the pelleted platelets were        re-suspended in 200 ml sterile docked saline.    -   5. The washed and re-suspended platelets were frozen at −20 to        −30° C. The freezing step was carried out within 4 hours from        the previous step.

The above elaborated collection and washing procedures of theleukocyte-reduced platelets aphaeresis unit were carried out in a bloodcollection center (Rock River Valley Blood Center, Rockford, Ill./FDALicense #249). The final WAP bags were supplied for the experimentswithin two months from the date of preparation. The units were receivedfrozen on dry ice.

Prior to further processing the individuals frozen units (bags) werethawed and then pooled together or pooled while frozen and thawedtogether. The thawing procedure was carried out at 25° C. while stirringat 30 RPM using a stainless steel propeller connected to a RK1 overheadstirrer (Heidolph Instruments, Germany).

Growth Factors Recovery.

The concentrations of several growth factors in the samples weredetected and measured using specific commercial ELISA kits (Quantikineby R&D Systems, MN USA: Human TGF-β1 Cat. DB100B, Human FGF basic Cat.HSFB00D, Human VEGF Cat. DVE00. Human PDGF-AB Cat. DHD00B. Human PDGF-BBCat. DBB00, and Human EGF Cat. DEG00). In all the experiments below,growth factors content was measured in the lysate before loading thesample to the chromatography resin (pre-SDR material) and aftercollecting the sample from the resin (following solvent and detergentremoval; post-SDR material), and the percentage of recovery of thegrowth factor following S/D removal was calculated.

Cell Count.

3T3-Swiss Albino fibroblasts cells (ATCC, Cat. number CCL92) were grownas an adherent monolayer. Every 2-3 days, the flasks were examined forconfluence using a microscope.

The counting procedure was carried out as following:

-   -   The culture medium was aspirated.    -   The cell layer was briefly rinsed with 10 ml PBS to remove all        traces of serum.    -   5 ml of Trypsin-EDTA (0.05% and 0.02%, respectively) solution        were added per 175 cm2 flask and the cells were observed under        an inverted microscope until the cell layer was dislodged from        the plate surface (usually within 3 to 5 minutes).    -   10 ml of complete growth medium [DMEM; Biological Industries,        Israel; Cat. number 01-055-1A containing 4.5 gr/l glucose and        supplemented with 4 mM glutamine (Biological Industries, Israel;        Cat. number 03-020-1B), 10% fetal calf serum (FCS; HyClone. USA;        Cat. number SH30070.03), penicillin (100 U/ml)/streptomycin (0.1        mg/ml)/amphotericine (0.25 μg/ml) solution (P/S/A; Biological        Industries. Israel; Cat. number 03-033-1B)] were added, and the        floating cell clumps/clusters were dispersed by gently pipetting        up and down several times until a homogenous cell suspension was        obtained.    -   The cell suspension was transferred into a 50 ml tube and        centrifuged in a swinging bucket rotor at 1000×g. for 4 minutes        at room temperature.    -   The supernatant (containing the medium) was discarded and the        pellet (containing the cells) was re-suspended in 3-10 ml of        fresh complete growth medium as above.    -   10 μl of the cell suspension were loaded in the haemocytometer        for cell counting.        Proliferation Assay.        On Day 1:

The cells were counted (as elaborated above), diluted to a concentrationof 25,000 cells/ml with complete growth medium (GM) and 100 μl from thecell solution were seeded and allow to adhere in rows B-G of a 96-wellplate (a final cell concentration of 2500 cells per well). Rows A+H wereleft empty. The plates were incubated for 24 hours at 37° C. in awater-jacketed incubator with 5% CO₂.

On Day 2:

The growth medium was aspirated and the plate adhered cells were washedtwice with starvation medium [DMEM containing 4.5 gr/l glucosesupplemented with 4 mM glutamine, 1% MEM-EAGLE non-essential amino acids(Biological Industries, Israel; Cat. number 01-340-1B, 1% human serumalbumin (Plasbumin 25, Talecris Biotherapeutics, Germany) and P/S/A (inthe concentrations listed above)](SM) (about 100 μl/well each wash) andfresh 100 ul/well SM were added to all wells (rows A-H). The plates wereincubated for 24 hours at 37° C. in a water-jacketed incubator with 5%CO₂.

On Day 3:

10 μl of undiluted or serially diluted tested material/extract (preparedaccording to a given treatment) was added to a well (in threereplicates) and incubated for additional 48 hours at 37° C. in awater-jacketed incubator with 5% CO₂. Diluted samples were prepared byperforming 6 or 9 serial dilutions of 1:2 or 1:3 with starvation medium.

On Day 4:

10 μl of a cell proliferation measuring reagent WST-1 (RocheDiagnostics, Mannheim. Germany; Cat. number 11-644-807; this reagent isdesigned to be used for the non-radioactive, spectrophotometricquantification of cell proliferation, growth, viability andchemosensitivity in cell populations using a 96-well-plate format) wereadded to each well. After an additional incubation of 4 hours at 37° C.in a water-jacketed incubator with 5% CO₂, the 96-well plate was read at450 nm and 650 nm in an ELISA reader after blanking the instrument onthe blank wells (rows A+H), containing the medium only.

Evaluation of Results.

The results obtained by ELISA reader at 650 nm were subtracted from theresults obtained at 450 nm per each well separately. The values werefurther analyzed by Prism software (GraphPad Software, Inc). To reducethe background reading, the results obtained for the untreated wells(containing cell that were not treated with the test materials) weresubtracted from all the values of wells on the same 96-wells plate. Theobtained results were used to plot a sigmoidal dose response curveagainst the log of the material concentration. In addition, R² fit,median effective concentration (EC50), and 95% Confidence Intervals EC50values were calculated by GraphPad Prism software.

Thrombin Activity.

Thrombin activity was assessed by clotting time measurements usingSTart4 Coagulation Instrument (Diagnostica Stago, Asnières sur Seine,France). The assay is a modification of the European Pharmacopoeia Assayprocedure, 1997, 0903, p. 858. Briefly, a calibration curve was preparedby mixing thrombin standard with a fibrinogen solution of 0.1%fibrinogen content (Enzyme Research Laboratories, IN, USA). Thrombinconcentration in the different tested extract samples is then calculatedfrom the calibration curve by their clotting time (the concentration isinterpolated from the calibration curve). Prior to the measurements thetested extract samples were mixed 1:1 (w/w) with thrombin 16 IU/ml(Omrix, Israel) to reach a final concentration of 8 IU thrombin/ml. Forthe calibration curve, a 8 IU thrombin/ml standard sample was preparedby mixing the same 16 IU thrombin/ml solution 1:1 (w/w) with thrombindilution buffer (0.4% tri-sodium citrate di-hydrate, 0.9% sodiumchloride and 1% BSA, pH=7.5). A positive control sample for thetreatment was made by mixing the same 16 IU thrombin i/ml solution 1:1(w/w) with a platelet extract sample which was not S/D treated and didnot contain heparin, LMWH or PVP (prepared as in Example 1). Thrombinactivity was measured in each lysate prior to and after the column. Theresults are shown relative to the control sample (considered as 100%thrombin activity).

PVPs used in the experiments below.

In Table 30 below the concentration w/w of different PVPs and theircorresponding concentration in mM are shown.

PVP is characterized by its K-value, or the Fikentscher's viscositycoefficient, which is a function of the average molecular weight, thedegree of polymerization, and the intrinsic viscosity. The averagemolecular weight of the soluble Kollidon grades is expressed in terms ofthe K-value in the pharmacopoeias valid in Europe and the USA.

PVP polymer is made by a polymerization reaction in which monomers arejoined together. Different molecular weights of PVP are made bycontrolling the termination of the polymerization reaction. This givesrise to the different types of PVPs, each with its own range of MW.There are several methods to determine the MW of PVP. Of note, theK-value and viscosity are not interchangeable. K-value is calculatedfrom the viscosity in water.

In all the experiments below the listed percentages of PVP are w/w ormM. Also, the acetate/glycine/HSA percentages are calculated as w/w.

In all platelet lysate preparations below, the osmolarity of the pooledWAP was about 260-280 mOs [measured by using The Advanced™ MicroOsmometer Model 3300 (Advanced Instruments Inc, Norwood, Mass., USA)].In order to keep the osmolarity level as constant as possible throughoutthe process, buffer osmolarity was monitored and adjusted, if needed, tothat of the WAP starting material using NaCl (Sigma-Aldrich, St. Louis,Mo., USA).

Example 1: Platelet Extract Prepared from Pooled Washed AphaeresisPlatelets Leukocyte Reduced (WAP), Treated with S/D, Admixed withHeparin, and Subjected to S/D Removal

In the following example the effect of including non-fractionatedheparin [Heparin Sodium-Fresenius 5000 i.u./1 ml (injection) Bodene(PTY) Limited. South Africa] during S/D removal by hydrophobicinteraction chromatography (HIC) on the recovery of TGF-β1, PDGF-AB,PDGF-BB, bFGF, VEGF, and EGF was examined. Heparin was tested since itis known to bind some growth factors. Heparin has a wide range ofmolecular weight, and by non-fractionated heparin it is meant that therewas no isolation or selection of specific narrower range of molecularsize.

S/D removal was carried out using SDR HyperD solvent-detergent removalchromatography resin (Pall Corp). SDR HyperD is a chromatographicpacking made of silica beads in which the pore volume is filled with athree-dimensional cross-linked hydrophobic acrylic polymer. The SDRHyperD involves a mixed-mode adsorption of hydrophobic interaction andis associated with a molecular exclusion effect [Guerrier L et al.“Specific sorbent to remove solvent-detergent mixtures fromvirus-inactivated biological fluids”. J Chromatogr B Biomed Appl. 1995Feb. 3; 664(1):119-125].

2 ml of the resin were packed in a 1 cm diameter Bio-Rad column (smallscale experiment). The S/D treated platelet lysate samples were preparedusing 965-2328 g pooled washed apheresis platelets leukocyte reduced(WAP) obtained from 5-12 bags (each bag is obtained from one donor). 20mM sodium acetate, 10 mM glycine and 0.2% human serum albumin (HSA) W/W(from the final volume solution) were added into the pooled WAP. In thenext step, 1% Triton X-100 and 0.3% TnBP were added into the solution,and the solution was incubated and mixed (on a tube roller) at roomtemperature (22±2° C.) for 2 hours for platelet lysis and antivirustreatment. The stock lysate was then aliquoted (14 ml) into vials,frozen and stored at −80° C. until use. Prior to use, the aliquotedlysate was thawed in a 37° C. water bath, filtered through 5 μm syringefilter to remove any particulate matter and mixed on a roller mixer forat least 5 minutes.

The packed columns with the resin were washed, prior to loading the S/Dtreated lysate, with 10 ml Purified Water, and equilibrated with 10 mlacetate-glycine-HSA buffer pH 6.8-7.4 (abbreviated as “AGA”;concentration as above; In the experiments below all AGA used was at apH of 6.8-7.4) with or without heparin according to the lysate's buffer(AGA with or without heparin). In the next step, 10 ml of theS/D-treated lysate were loaded onto the column except for samples 2 and4 which were incubated with 5 IU heparin/ml (50 μl heparin was addedinto 50 ml of platelet lysate) prior to loading the lysate onto thecolumn. The flow rate was kept up to 0.4 ml/min. Prior to loading thecolumn, all samples with or without heparin, were mixed on a tube rollerfor 20 minutes at room temperature (22±2° C.). The residual S/D-treatedlysate material (which was not loaded onto the column) was transferredinto 1.5 ml vials and kept at −80° C. for analysis for growth factorconcentration measurements and was regarded as loading control/pre-SDRmaterial.

After loading the lysates, the columns were loaded with differentbuffers (as shown in the Table 1 below), 10 ml each. Each buffercontained different ingredients at different concentrations. In some ofthe buffers, heparin was combined with NaCl/ethanol. The flow rate waskept at or below 0.8 ml/min. All fractions obtained from the columnafter loading, and after washing with the buffers were collected,combined, and the collected material was divided into 1 ml aliquots andwas kept frozen under −80° C. until proceeding with the measurements ofgrowth factor recovery. This material was regarded as post-SDR removalmaterial.

The recovery of different growth factors (% relative to the pre-SDRmaterial) is shown in Table 2.

TABLE 1 A detailed description of samples and conditions used during theS/D removal step. Final volume of Treat- Sample post-SDR ment loadedBuffer 1 Buffer 2 Buffer 3 material (ml) 1 S/D AGA AGA + AGA + 40treated 12.5% 10% Lysate EtOH + EtOH + 0.5M 1M NaCl NaCl + 5 IU/mlheparin 2 S/D AGA AGA + AGA + 40 treated 12.5% 10% Lysate + EtOH +EtOH + 5 IU/ml 0.5M 1M NaCl Heparin NaCl + 5 IU/ml heparin 3 S/D AGA +AGA + 30 treated 12.5% 10% Lysate EtOH + EtOH + 0.5M 1M NaCl + NaCl 5IU/ml heparin 4 S/D AGA + AGA + 30 treated 12.5% 10% Lysate + EtOH +EtOH + 5 IU/ml 0.5M 1M Heparin NaCl + NaCl 5 IU/ml heparin

TABLE 2 Recovery of various growth factors in a platelet extractfollowing S/D removal step (% relative to pre-SDR column) according tothe samples and conditions in Table 1. Growth factor Recovery (%) PDGF-PDGF- Treatment AB BB bFGF VEGF EGF TGF-β1 1 50.4 62.6 52.6 55.8 54.976.1 2 49.1 77.3 92.9 62.4 50.3 77.3 3 61.7 86.0 53.7 66.7 66.9 76.2 460.6 92.1 97.0 50.2 56.7 66.9

The results presented in Table 2 show that incubation of an S/D-treatedplatelet lysate with heparin prior to loading the material to an SDRcolumn (treatments 2 and 4 vs. 1 and 3) resulted in a dramatic increasein the recovery of bFGF from about 53% to about 93 or 97%. The recoveryof PDGF-BB also increased, but to a lesser extent. Treatments 2 and 4,which included a combination of incubation with heparin prior to loadingto the SDR column and an additional washing step with heparin resultedin a significant enrichment of the resulting platelet extract withPDGF-AB and PDGF-BB which is further increased in treatment 4 (61% and92% recovery, respectively) by washing the column, immediately afterloading, with a buffer containing heparin as opposed to treatment 2 inwhich preceding the use of the heparin containing buffer, the column iswashed with a buffer without heparin.

Therefore, the results show that contacting the S/D treated plateletlysate with heparin prior to the removal of the S/D, e.g. by HIC, canadvantageously increase the recovery of certain growth factors. e.g.bFGF and PDGF-BB.

Example 2: The Effect of Platelet Extract Prepared by Admixing theLysate with Heparin Prior to S/D Removal on Fibroblast CellProliferation

In the following example the biological effect of a platelet extractprepared by contacting the lysate with heparin prior to S/D removal on3T3-Swiss Albino fibroblast cell proliferation was examined. Fourextracts prepared according to the different four steps elaborated inTable 1 were examined. The results are shown in FIG. 1.

The cell proliferation results (FIG. 1) show that samples obtained bytreatments 2 and 4 including incubation with heparin prior to loading onthe SDR column were significantly more effective in inducingproliferation than samples obtained by treatments 1 and 3 that were notincubated with heparin. Samples obtained by treatment 2 and 4 had EC50of 0.99 and 0.89, respectively, whereas samples obtained by treatments 1and 3 had EC50 of 2.93 and 2.4, respectively.

These results demonstrate that incubation with heparin prior to S/Dremoval, which resulted in higher GFs recovery, also improved biologicalpotency as reflected by the fibroblasts proliferation assay.

Example 3: The Effect of Admixing an S/D-Treated Lysate with LowMolecular Weight Heparin Prior to S/D Removal Step

Low molecular weight heparins (LMWHs) are heparins having an averagemolecular weight of about 4.5 kDa compared to 15 kDa of anunfractionated heparin. In the clinical setting, LMWH has severalpharmacological and practical advantages over unfractionated heparin.LMWH have been shown to have less unspecific binding to cells andproteins, has a longer plasma half-life, and can therefore beadministered subcutaneously (Hirsh and Raschke, Heparin andlow-molecular-weight heparin: the Seventh ACCP Conference onAntithrombotic and Thrombolytic Therapy. Chest. 2004; 126:188S-203S).

In the following example the effect of different conditions used duringthe HIC S/D removal step on the recovery of PDGF-AB, PDGF-BB, bFGF,VEGF, and EGF were examined (see the different conditions in Table 3).In this experiment, Enoxaparin sodium (Clexane, Sanofi Aventis) was usedas a low molecular weight heparin.

The lysates were prepared and then loaded to an SDR column as elaboratedin the experiment above under the conditions elaborated in Table 3.Prior to loading, equilibration was carried out using the buffer of theloaded lysate. All fractions obtained from the column after loading, andafter washing with the buffers were collected, combined, and the totalgrowth factors recovery was calculated. Treatment 2 included incubationwith 5 IU/ml Enoxaparin in the same manner as was carried out forheparin above. The results are shown in Table 4.

TABLE 3 A detailed description of samples and conditions used during theS/D removal step. Final volume of post- Treat- Sample SDR ment loadedBuffer 1 Buffer 2 Buffer 3 material 1 S/D treated AGA AGA + AGA + 40lysate 12.5% 10% EtOH + EtOH + 0.5M 1M NaCl NaCl + 5 IU/ml Enoxaparin 2S/D treated AGA + AGA + 30 lysate + 12.5% 10% 5 IU/ml EtOH + EtOH +Enoxaparin 0.5M 1M NaCl + NaCl + 5 IU/ml 5 IU/ml Enoxaparin Enoxaparin

TABLE 4 Recovery of various growth factors in a platelet extractfollowing S/D removal step (% relative to pre-SDR column) according tothe samples and conditions in Table 3. Growth factor Recovery (%)Treatment PDGF-AB PDGF-BB bFGF VEGF EGF 1 38.8 63.9 43.0 53.4 63.7 255.9 74.9 61.5 58.2 60.4

The results presented in Table 4 show that incubation of the S/D-treatedplatelet lysate with Enoxaparin prior to S/D removal step (treatment 2vs. 1), and an additional Enoxaparin-containing washing step increasedthe recovery of PDGF-AB, PDGF-BB and bFGF.

However, the recoveries following contact with 5 IU/ml unfractionatedheparin (treatment 4 in Table 1; Example 1 above) for PDGF-BB and bFGF(92% and 97%, respectively) were higher than with 5 IU/ml Enoxaparin(75% and 62%, respectively). An additional experiment with increasedEnoxaparin concentration (30 IU/ml; data not shown), did not result inany significant change in growth factor recoveries.

Example 4: The Effect of Platelet Extract Prepared by Admixing theLysate with Low Molecular Weight Heparin Prior to S/D Removal onFibroblast Cell Proliferation

In the following example the effect of a platelet extract prepared asdescribed in Example 3 on cell proliferation was examined. Cell countand proliferation assay was carried out as elaborated in the “MATERIALSand METHODS” section using 3T3-Swiss albino fibroblast cells. Theresults are shown in FIG. 2.

The results (FIG. 2) show that fibroblasts proliferation was higher inthe sample that was incubated with LMWH (sample 2/treatment 2, EC500.05) compared with the sample that was not incubated with LMWH (sample1/treatment 1, EC50 0.11).

These results demonstrate that incubation with low molecular weightheparin prior to S/D removal increases the recovery of some growthfactors (Example 3) and also improves the biological potency.

Example 5: The Effect of Admixing a Platelet Lysate with PVP Prior toS/D Removal on Growth Factor Recovery

In the previous Example, prior to S/D removal, the lysate was contactedwith unfractionated heparin and LMWH, which are known to bind certaingrowth factors.

In the following Example, PVP, a completely different compound havingamphiphilic characteristics, was explored on its effect on growth factorrecovery following S/D removal by HIC column.

In this example different S/D removal conditions were tested with orwithout (control) the addition of PVP prior to or during the HIC S/Dremoval step. The recovery of the following growth factors was examined:TGF-β1, PDGF-AB, PDGF-BB, bFGF, VEGF, and EGF using the commercial ELISAkits listed above. Two types of PVP were tested: K25 (Kollidon® 25having a K-Value of 22.5-27.0 and an average molecular weight of 30000Da, Cat. 02286 Sigma Life Sciences, Germany); and K30 (Povidone K-30having a K-Value of 27.0-32.4 and an average molecular weight of 40000Da, Cat. P1454 Spectrum chemical mfg corp. USA).

The lysates were prepared and loaded onto a SDR column as elaborated inExample 1 above and by using the S/D removal conditions as elaborated inTable 5. Prior to loading, equilibration was carried out using thebuffer of the loaded lysate. All fractions obtained from the columnafter loading, and after washing with buffer 1 and buffer 2 werecollected, combined, and the growth factor recovery was calculated.Growth factor recovery results (calculated as explained above) are shownin Table 6. All loaded samples that comprised PVP were also incubatedwith PVP in the manner discussed above.

TABLE 5 A detailed description of samples and conditions used during theS/D removal step. Treatment Sample loaded Buffer 1 Buffer 2 7 S/Dtreated AGA + 0.1% AGA + 1% lysate + 1% (0.025 mM) PVP 30 (0.25 mM) PVP30 + 0.5M PVP 30 NaCl + 12.5% EtOH 3 S/D treated AGA + 0.1% AGA + 1%lysate + 1% (0.03 mM) PVP 25 (0.3 mM) PVP 25 + 0.5M PVP 25 NaCl + 12.5%EtOH 6 S/D treated AGA + 0.1% AGA + 0.1% lysate + 0.1% PVP 25 PVP 25 PVP25 8 S/D treated AGA + 0.1% AGA + 0.1% lysate + 0.1% PVP 30 + PVP 30 +PVP 30 0.5M NaCl + 1M NaCl + 12.5% EtOH 12.5% EtOH 9 S/D treated AGA +0.1% AGA + 1% lysate + 1% PVP 30 PVP 30 PVP 30 5 S/D treated AGA + 0.1%AGA + 1% lysate + 1% PVP 25 PVP 25 PVP 25 2 S/D treated AGA + 0.5M AGA +1M lysate NaCl + 12.5% NaCl + EtOH 12.5% EtOH 4 S/D treated AGA + 0.1%AGA + 0.1% lysate + 0.1% PVP 25 + PVP 25 + PVP 25 0.5M NaCl + 1M NaCl +12.5% EtOH 12.5% EtOH 10 S/D treated AGA + 0.1% AGA + 0.1% lysate + 0.1%PVP 30 PVP 30 PVP 30 1 S/D treated AGA + 0.5M AGA lysate NaCl + 12.5%EtOH

TABLE 6 Recovery of various growth factors in a platelet extractfollowing S/D removal step (% relative to pre-SDR column) according tothe samples and conditions in Table 5. Growth factor Recovery (%) PDGF-PDGF- Treatment AB BB bFGF EGF VEGF 7 90 90 100 65 81 3 83 86 88 67 72 680 94 59 59 60 8 70 87 66 57 49 9 58 34 89 69 82 5 47 29 83 67 82 2 4865 28 59 51 4 29 30 35 65 79 10 41 45 53 62 63 1 49 45 20 50 55

The results show that contacting an S/D-treated platelet lysate with PVPprior to and during S/D removal by an SDR column greatly affected therecovery of growth factors from an SDR column.

Also, the results show that, during the S/D removal step, PVP combinedwith ethanol and NaCl was more effective in increasing growth factorrecovery than PVP alone (compare treatments 7 vs. 9, and 3 vs. 5).

The results also show that using a lower molecular weight PVP resultedin a further increase in growth factor recovery (compare treatment 3with 7).

It can be concluded that an increase in growth factor recovery, whensubjecting an S/D treated lysate to an S/D removal step, can be achievedby contacting the S/D treated lysate with PVP prior to and/or during theS/D removal step.

Example 6: The Effect of the Molecular Weight of the PVP Polymer UsedPrior to and During S/D Removal on Fibroblast Cell Proliferation

In the following example the effect of the molecular weight of the PVPcontacted with an S/D treated lysate prior to and during S/D removal oncell proliferation was examined.

Platelet lysate samples prepared according to the S/D removal procedureelaborated for treatments 3 and 7 in Table 5 (Example 5) were used.

Cell count and cell proliferation assay were carried out as elaboratedin the “MATERIALS and METHODS” section using 3T3-Swiss albino fibroblastcells.

The results are shown in FIG. 3.

The results show that sample 3 comprising PVP K25 had a higherproliferation rate (EC50=0.34) than sample 7 comprising PVP K30(EC50=1.95). Although, as indicated in Example 5, the recovery of growthfactors resulting from treatment 7 was higher than the recovery ofgrowth factors resulting from treatment 3, the activity of the growthfactors recovered in treatment 3 is much more effective than theactivity of the growth factors recovered in treatment 7.

These results suggest that in order to obtain an increased biologicalpotency, a lower molecular weight of PVP (e.g. PVP K25) can beadvantageously used.

Example 7: The Effect of Different Concentrations of PVP Prior to andDuring S/D Removal on Growth Factor Recovery

The following example aims to corroborate the previous results showingthat using PVP, ethanol and/or NaCl during S/D removal step increasesgrowth factor recovery from an SDR column. The effect of differentconditions during the S/D removal step was examined and the recovery ofseveral growth factors in the post-SDR material was measured asspecified above (TGF-β1, PDGF-AB, PDGF-BB, bFGF, VEGF, and EGF).

PVP K25 (same as above) was used in these experiments. The lysates wereprepared and loaded onto a SDR column as elaborated above in Example 1using the S/D removal conditions as elaborated in Table 7 below. Priorto loading, equilibration was carried out with AGA (concentrations asabove)+0.1% (0.03 mM) PVP K25. All samples contained a finalconcentration of 0.1% (0.03 mM) PVP K25 in AGA buffer (concentrations asabove). All loaded samples were incubated with PVP in the mannerdiscussed above before loading. All fractions obtained from the columnafter loading, and after washing with the buffers were collected,combined, and the growth factor recovery was calculated. The growthfactor recovery results (% relative to the pre-SDR material) arepresented in Table 8 and are listed from the highest to the lowest totalgrowth factor recovery.

One aim was to try to reduce the PVP in the final product. In someexperiments described in the preceding Examples, the sample wasincubated with a relatively high PVP final concentration of 1% (Example5). In some cases, after loading the column with the sample volume (6column volumes) the column was washed with a relatively low PVPconcentration of 0.1%. However, since the largest fraction by volume isthe sample loaded (6 column volumes), the best way to reduce the PVP inthe product is by reducing PVP concentration in the sample to be loadede.g. by incubating the sample to be loaded with a final concentration of0.1% (0.03 mM) PVP K25 instead of 1% (0.3 mM) PVP K25.

TABLE 7 A detailed description of samples and conditions used during theS/D removal step. Treatment Buffer 1 Buffer 2 19 AGA + 0.5% PVP AGA +0.5% PVP + 1M NaCl + 12.5% EtOH 18 AGA + 0.5% PVP + AGA + 0.1% PVP + 1M0.5M NaCl + 12.5% NaCl + 12.5% EtOH EtOH 13 AGA + 1% PVP AGA + 0.1%PVP + 1M NaCl + 12.5% EtOH 17 AGA + 0.5% PVP AGA + 0.1% PVP + 1M NaCl +12.5% EtOH 8 AGA + 1% PVP + AGA 1M NaCl + 12.5% EtOH 7 AGA AGA + 1%PVP + 1M NaCl + 12.5% EtOH 20 AGA + 0.5% PVP + AGA + 0.5% PVP + 1M 0.5MNaCl + 12.5% NaCl + 12.5% EtOH EtOH 9 AGA + 0.1% PVP AGA + 1% PVP + 1MNaCl + 12.5% EtOH 2 AGA + 0.1% PVP AGA + 0.1% PVP + 1M NaCl + 12.5% EtOH3 AGA + 0.1% PVP + AGA + 0.1% PVP + 1M 0.5M NaCl + 12.5% NaCl + 12.5%EtOH EtOH 12 AGA + 0.1% PVP + AGA + 1% PVP 1M NaCl + 12.5% EtOH 6 AGA +0.5M NaCl + AGA + 1% PVP + 1M 12.5% EtOH NaCl + 12.5% EtOH 11 AGA + 0.1%PVP + AGA + 1% PVP + 1M 1M NaCl + 12.5% NaCl + 12.5% EtOH EtOH 10 AGA +0.1% PVP + AGA + 1% PVP + 1M 0.5M NaCl + 12.5% NaCl + 12.5% EtOH EtOH 14AGA + 0.5% PVP AGA + 0.5% PVP 16 AGA + 0.5% PVP AGA + 0.1% PVP 4 AGAAGA + 1% PVP 5 AGA + 1% PVP AGA 15 AGA + 0.1% PVP AGA + 0.5% PVP 1 AGA +0.1% PVP AGA + 0.1% PVP

TABLE 8 Recovery of various growth factors in a platelet extractfollowing S/D removal step according to the samples and conditions inTable 7. Growth factor Recovery (%)* Treatment PDGF-AB bFGF VEGF EGFTGF-β1 19 76 73 75 60 74 18 89 74 50 57 83 13 75 56 49 64 101 17 71 6572 63 70 8 74 71 71 55 65 7 72 60 67 57 74 20 73 56 50 54 88 9 61 68 6461 67 2 56 60 73 60 65 3 62 60 53 54 83 12 60 48 51 57 93 6 65 61 53 5474 11 64 58 53 53 75 10 54 69 50 57 73 14 25 32 87 54 98 16 18 40 77 6098 4 21 29 76 65 90 5 19 32 72 64 88 15 13 26 71 60 98 1 15 25 69 59 86*% relative to pre-SDR column.

The results confirm the previous results and show that the highestgrowth factor recovery from the SDR column was obtained when the lysatewas contacted with PVP in combination with ethanol and NaCl than whencontacted with PVP alone during the S/D removal step.

The results also show that it is possible to reduce the PVPconcentration in the product to below 0.5% (0.17 mM).

Example 8: The Effect of Different Concentrations of PVP in a PlateletExtract on Fibroblast Cell Proliferation

The proliferative effect of samples prepared in Example 7 on cellproliferation was examined in the manner described in the “MATERIALS andMETHODS” section using 3T3-Swiss albino fibroblasts cells.

In this experiment, cell proliferation activities of sample 17 and 19,which showed comparable amounts of growth factor recovery, wereexamined. The main difference between the two samples was that sample 17was prepared by a second washing buffer comprising 0.1% (0.03 mM) PVPwhereas sample 19 was prepared with a second washing buffer comprising0.5% (0.17 mM) PVP (see Table 7). This difference resulted in twoextract products having different PVP concentration.

The results show that cell proliferation activity in sample 17 whichcomprises a low PVP concentration (prepared with 0.1% PVP; EC50=2.3) wassimilar to that of sample 19 (prepared with 0.5% PVP; EC50 2.87) whichcomprises a higher PVP concentration.

The results (FIG. 4) show that higher amount of PVP in the product didnot affect proliferation level.

Example 9: The Effect of PVP on Thrombin Activity

The following example was aimed to examine whether a platelet extractprepared using PVP or heparin during S/D removal affects thrombinactivity.

Thrombin is a key enzyme of coagulation and is activated as the firststep of the coagulation cascade. Thrombin acts as a serine protease thatconverts soluble fibrinogen into insoluble strands of fibrin, as well ascatalyzes many other coagulation-related reactions.

The effect of heparin and LMWH on blood coagulation has been known andit is used principally in medicine for anticoagulation [Machovich R etal. “Effect of Heparin on Thrombin Inactivation by Antithrombin-III”.Biochem. J. 1978; 173:869-875].

Thrombin activity was assessed by the clotting time measurement asdescribed above. The results shown in Table 9 are relative to thecontrol sample (which is considered as 100% thrombin activity).

TABLE 9 Thrombin activity (% relative to control sample) of plateletextract samples comprising heparin, LMWH or PVP 25. LMWH-comprising PVPK25-comprising Heparin-comprising platelet platelet extract plateletextract sample** extract sample*** sample**** pre- post- pre- post-SDRpost-SDR SDR SDR material SDR material pre- material material Treatment2 Treatment 4 material Treatment 2 SDR material Treatment 17 62* 52* 61*96* 86* 100* 100* *Thrombin activity (%) relative to control sample.**Prepared according to treatment 2 and 4 in Example 1. ***Preparedaccording to treatment 2 in Example 3. ****Prepared according totreatment 17 in Example 7.

The results show that platelet extract samples comprising unfractionatedheparin or LMWH had an inhibitory effect on thrombin activity in vitro,whereas no inhibitory effect was detected with platelet extract samplescomprising PVP.

Example 10: Platelet Extract Prepared from Pooled Washed AphaeresisPlatelets Leukocyte Reduced (WAP), Treated with S/D, Incubated with PVPK25, and Washed from a SDR Column with PVP K25 Prepared in a Large ScaleProcess

In this experiment, the effect of PVP addition prior S/D removal on therecovery of growth factor was evaluated in higher scale (in the aboveexperiments a small scale process was carried out). In this experimentPVP K25 was used.

The lysate was prepared as follows: Platelet lysate samples wereprepared using 2328 g pooled washed apheresis platelet leukocyte reduced(WAP) obtained from 12 bags. 255 ml acetate-glycine buffer to a finalconcentration of 20 mM sodium acetate, 10 mM glycine; at pH 6.8-7.4 andHuman Serum Albumin (HSA; Talecris USA) to a final concentration of 0.2%W/W from the final volume solution were added into the pooled WAP. Inthe next step, S/D treatment was carried out by slowly adding 1% TritonX-100 and 0.3% TnBP (w/w) into the pooled sample while mixing at 30 RPM.In order to avoid sub-optimal viral inactivation due to the possiblepresence of particulate matter, the S/D treatment was split into twoparts. First, the sample was continuously stirred for 30 minutes,centrifuged at 5016×g for 10 minutes at 23-27° C., and filtered through0.45 μm filter (Sartopore 2, Sartorius Stedim Biotech S.A., Aubagne,France). In the second part, the filtered material was poured into astainless steel pot immersed in a water bath adjusted to 25° C. andmixed at 30 RPM for additional 2 hours to continue the viralinactivation process in a clear solution. PVP K25 was added to theS/D-treated lysate to a final concentration of 0.1% (w/w) (0.03 mM) andincubated for 20 minutes at 25° C. while stirring at 30 RPM. The samplewas filtered using 5 μm Sartopore PP2 filter to remove particulatematter.

Next, S/D removal was carried out using XK50 liquid chromatographycolumn packed with 300 ml SDR HyperD solvent-detergent removalchromatography resin (Pall Corp) in conjunction with a peristaltic pumpand a UA-6 UW/WIS detector+Type 11 recorder (ISCO, NE, USA). The columnwas equilibrated with 900 ml of Acetate-Glycine buffer containing 20 mMNa-acetate, 10 mM glycine, 0.1% (0.03 mM) PVP and 0.2% HSA, pH 6.8-7.4.1800 ml of S/D- and PVP-treated platelet lysate (which contained 1620 mlof platelet material) were loaded onto the column followed by washingthe column with 600 ml acetate glycine buffer (same concentrations asabove) containing 0.5% (0.17 mM) PVP and 0.2% HSA. This was followed bywashing with 1,200 ml of Acetate-Glycine buffer containing 12.5%ethanol, 1M NaCl, 0.1% (0.03 mM) PVP and 0.2% HSA. Next, the column waswashed with 300 ml of purified water. The extract included all fractionscombined and collected from the column after loading, and after washingwith the buffers. Growth factor recovery in the extract was measured andcalculated.

A total volume of 3600 ml was collected from the column. The collectedmaterial was filtered using consecutively 3 and 1.2 μm Sartopure PP2filters and a 0.45 μm Sartopore 2 filter (Sartorius Stedim Biotech S.A.,Aubagne, France).

PDGF-AB, PDGF-BB, VEGF, TGF-β1, bFGF and EGF recoveries were calculatedas described above.

TABLE 10 Recovery of the various growth factors in a platelet extractfollowing S/D removal step according to the above conditions in a largescale process. Growth factor Recovery (%)* TGF-β1 83 PDGF-AB 73 PDGF-BB87 EGF 59 bFGF 61 VEGF 76 *% relative to pre-SDR column.

The results presented in Table 10 show that the relatively high growthfactor recoveries from the SDR column when using low concentration ofPVP in combination with ethanol and NaCl are maintained when carryingout a large process scale. The concentration of PVP in the post-SDRextract was 0.17% (0.057 mM).

Example 11: The Effect of a Platelet Extract Prepared in a Large ScaleProcess by Contacting the Lysate with Low Concentration of PVP K25 Priorto and During S/D Removal on Fibroblast Cell Proliferation

The effect of a sample prepared in the previous Example with PVP K25 oncell proliferation was carried out as elaborated above using 3T3-Swissalbino fibroblasts cells. The activity of the sample (marked astreatment 1 in FIG. 5) was compared to the activity of a lysate preparedby contact with heparin as elaborated in Example 1 (marked as treatment2 in FIG. 5).

The proliferation results are shown in FIG. 5.

The results show that platelet extract samples prepared in a large scaleprocess had a positive proliferative effect (EC50=0.024) which is higherthan the proliferative effect of a platelet extract sample prepared withheparin (EC50=0.047).

These results demonstrate that a platelet extract collected from a largescale SDR column as disclosed has a high growth factor recovery and thushas a biological activity when carried out in a large process scale.

Example 12: The Effect of Using PVP Prior to and/or During S/D MaterialRemoval on the Residual Levels of S/D Material in the Post-SDR Material

In the previous set of experiments it was shown that increased growthfactor recovery was obtained while contacting the platelet lysate withPVP, ethanol and/or NaCl prior to and/or during the S/D removal step.

It is important to verify that PVP does not reduce the S/D bindingcapacity of the SDR resin, to ensure efficient S/D removal from thelysate.

In the following set of experiments, the efficacy of S/D (TritonX-100and TnBP) removal under the same conditions as elaborated in Example 10was evaluated.

Of note, the acceptable limit of both Triton X-100 and TnBP inblood-derived products is <5 ppm. The concentration of Triton X-100 andTnBP was measured prior (pre-SDR material) to and following (post-SDRmaterial) the S/D removal step. Triton X-100 was determined by reversedphase HPLC with a U.V. detector, and TnBP was determined by capillarygas chromatography using a Flame Ionization Detector.

The results are shown in Table 11 below.

TABLE 11 S/D concentration in the lysate prior to and following S/Dremoval according to the conditions elaborated in Example 10. MaterialS/D ppm pre-SDR material Triton X-100 9,136 TnBP 2,586 post-SDR materialTriton X-100 <5 TnBP <0.3

In the above experiment, incubation/equilibration/washing were carriedout in the presence of PVP K25 in a concentration of up to 0.5% (0.17mM).

The results show that carrying out an S/D material removal in thepresence of PVP K25 in a concentration of up to 0.5%, which was found tobe efficient for growth factor recovery in the previous experiments, didnot affect the S/D removal performance of the column.

Additional experiments (carried out in small scale process as shown inExample 5), wherein incubation/equilibration/washing were carried out inthe presence of PVP K25 and K30 at a concentration of 1% (0.3 mM)resulted in the presence of Triton X-100 in the post-SDR. The resultsare shown in Table 12 below.

TABLE 12 S/D concentration in the lysate prior to and following S/Dremoval according to the conditions elaborated in Example 5. TreatmentMaterial S/D ppm 3 pre-SDR material Triton X-100 8587 TnBP 2480 post-SDRmaterial Triton X-100 60.8 TnBP <0.3 5 pre-SDR material Triton X-1008587 TnBP 2480 post-SDR material Triton X-100 7.2 TnBP <0.3 7 pre-SDRmaterial Triton X-100 8142 TnBP 2437 post-SDR material Triton X-100 7.9TnBP <0.3 9 pre-SDR material Triton X-100 8142 TnBP 2437 post-SDRmaterial Triton X-100 14.9 TnBP <0.3

It was concluded that, advantageously, carrying out an S/D removal inthe presence of PVP K25 in a concentration of lower than 1% (0.3 mM)results in increased growth factor recovery during S/D removal step andat the same time ensures efficient S/D removal from the lysate.

Example 13: The Ratio Between Several Growth Factors in a PlateletExtract Prepared from WAP, Treated with S/D, Contacted with PVP K25, andSubjected to S/D Removal

The following Example shows the ratio between several growth factors ina platelet extract prepared as disclosed, and examines whether theobtained ratio is comparable to that in the starting material, and tothat in the lysate before loading the sample onto the chromatographyresin (the pre-SDR material). The pre- and post-SDR material (orextract) were prepared as described in Example 10.

The levels of TGF-β1, VEGF, bFGF, and PDGF-AB were measured in all threetested materials (WAP starting material, pre-SDR material, and post-SDRmaterial) using the specific commercial ELISA kit described above, andthe ratios between PDGF-AB/TGF-β1; PDGF-AB/VEGF; TGF-β1/bFGF; andVEGF/bFGF were calculated. The growth factors levels and ratios areshown in Table 13 and 14, respectively, below.

TABLE 13 Levels of TGF-β1, VEGF, bFGF, and PDGF-AB in WAP startingmaterial, pre-SDR material, and post-SDR material. Growth factor level(ng) Tested material TGF-β1 VEGF bFGF PDGF-AB WAP starting material301445 2965 267 119520 Pre-SDR material 299228 2396 240 102308 Post-SDRmaterial 249563 1818 147 74365

TABLE 14 Calculated growth factor ratio in WAP starting material,pre-SDR material, and post-SDR material. Tested PDGF-AB/ PDGF-AB/TGF-β1/ VEGF/ material TGF-β1 VEGF bFGF bFGF WAP starting 0.40 40 112911 material pre-SDR 0.34 43 1247 10 material post-SDR 0.30 41 1698 12.4material

The results show that a platelet lysate contacted with PVP K25 incombination with ethanol and NaCl during a S/D removal step results inan extract having PDGF-AB/TGF-β1; PDGF-AB/VEGF; TGF-β1/bFGF; andVEGF/bFGF ratios which are similar to the ratios in the startingmaterial and in the material prior to S/D removal.

It can be concluded that carrying out an S/D removal as disclosedresults in a platelet extract comprising a proportion of factors that issimilar to the material before S/D removal.

Example 14: Growth Factor Recovery in Platelet Extracts Prepared byContacting the Lysate with Heparin, Dextran Sulfate or PVP K25 Prior toand/or During the S/D Removal Step

The following Example compares the growth factor recovery in differentplatelet extracts prepared by contacting the lysate with heparin,dextran sulfate or PVP during the S/D removal step. The recovery wascalculated as elaborated above. A platelet extract with heparin wasprepared using 1900-2500 g pooled washed apheresis platelets leukocytereduced (WAP) obtained from 10-13 bags. 209-385 ml acetate-glycinebuffer was added to a final concentration of 20 mM sodium acetate, 10 mMglycine; at pH 6.8-7.4 and 0.2% w/w (final concentration) Human serumalbumin (HSA, Talecris USA) were added into the pooled WAP. S/Dtreatment was carried out by slowly adding 1% Triton X-100 and 0.3% TnBP(w/w) into the pooled sample while mixing at 50 RPM. First, the samplewas continuously stirred for 30 minutes, and then filtered consecutivelythrough 20 and 3 μm Sartopure PP2 filters and 0.45 μm Sartopore 2(Sartorius Stedim Biotech S.A., Aubagne, France). Then, the filteredmaterial was returned to a beaker immersed in a water bath adjusted to25° C. and mixed at 50 RPM for additional 2 hours for continuing theviral inactivation process. S/D removal was carried out using XK50liquid chromatography column packed with 295 ml SDR HyperDsolvent-detergent removal chromatography resin (Pall Corp) inconjunction with a peristaltic pump and a UA-6 UW/WIS detector+Type 11recorder (ISCO, NE, USA). Equilibration was carried out with therespective buffer of the loaded sample. 1800 ml of S/D-treated plateletlysate (which contained 1620 ml of platelet material) were loaded ontothe column followed by washing with 600 ml acetate glycine buffer (sameconcentrations as above)+0.2% HSA. 600 ml of acetate glycine buffer(same concentrations as above) containing 12.5% ethanol, 0.5M NaCl, 5IU/ml Heparin (Heparin Sodium-Fresenium 5000 IU/ml. Bodene (PTY) Ltd,South Africa) and 0.2% HSA. This was followed by a second washing stepcarried out with 600 ml of acetate glycine buffer (same concentrationsas above) containing 10% ethanol, 1M NaCl and 0.2% HSA. The column wasfinally washed with 300 ml of purified water. The flow-through and allfractions obtained in the washing steps were collected and pooled (about3.6 liter). The collected material was filtered using consecutively 3and 1.2 μm Sartopure PP2 filters and 0.45 μm Sartopore 2 (SartoriusStedim Biotech S.A., Aubagne, France).

A platelet extract with dextran sulfate was prepared using 1800-2050 gpooled washed apheresis platelets leukocyte reduced (WAP) obtained from10 bags. 197-224 ml acetate-glycine buffer was added to a finalconcentration of 20 mM sodium acetate, 10 mM glycine; at pH 6.8-7.4 and0.2% w/w (from the final volume solution) Human serum albumin (HSA,Talecris USA) were added into the pooled WAP. S/D treatment was carriedout by slowly adding 1% Triton X-100 and 0.3% TnBP (w/w) into the pooledsample while mixing at 50 RPM. In order to avoid sub-optimal viralinactivation due to the possible presence of particulate matter, the S/Dtreatment was split into two parts. First, the sample was continuouslystirred for 30 minutes and then filtered through 20 and 3 μm SartopurePP2 filters and 0.45 μm Sartopore 2 (Sartorius Stedim Biotech S.A.,Aubagne, France). Then, the filtered material was returned to a beaker,immersed in a water bath adjusted to 25° C. and mixed at 50 RPM foradditional 2 hours for continuing the viral inactivation process.

Dextran sulfate (Sigma-Aldrich. Canada; Cat. number D4911) was added tothe sample to a final concentration of 1% (w/w) and incubated at 25° C.while stirring at 50 RPM for 20 minutes. The sample was filtered using 5μm Sartopore PP2 filter to remove particulate matter.

Next, S/D removal was carried out using XK50 liquid chromatographycolumn packed with 295 ml SDR HyperD solvent-detergent removalchromatography resin (Pall Corp) in conjunction with a peristaltic pumpand a UA-6 UW/WIS detector+Type 11 recorder (ISCO, NE, USA). The columnwas equilibrated with 900 ml of acetate glycine buffer containing 1%dextran sulfate and 0.2% HSA. 1800 ml of S/D- and dextransulfate-treated platelet lysate (which contained 1620 ml of plateletmaterial) were loaded onto the column followed by washing with 600 mlacetate-glycine buffer (same concentrations as above) with 12.5%ethanol, 0.5M NaCl, 0.1% dextran sulfate and 0.2% HSA. This was followedby washing with 600 ml of acetate-glycine buffer (same concentrations asabove) containing 1% dextran sulfate and 0.2% HSA. Next, the column waswashed with 300 ml of purified water. A total volume of 3000 ml wascollected from the column. The collected material was filtered using 3and 1.2 μm Sartopure PP2 filters and 0.45 μm Sartopore 2 (SartoriusStedim Biotech S.A., Aubagne, France).

A platelet extract with PVP was prepared as elaborated in Example 10.

The comparative growth factor recovery is shown in Table 15 below.

TABLE 15 Growth factor recovery of the different extracts. Growth factorRecovery (%)*** Extract-Dextran Extract-Heparin Sulfate Extract-PVP(Average ± SD)* (Average ± SD)** K25 TGF- β1 91 ± 8 74 ± 5  83 PDGF-AB43 ± 5 61 ± 10 73 PDGF-BB 69 ± 9 65 ± 5  87 bFGF  56 ± 10 44 ± 10 61 EGF56 ± 3 52 ± 5  59 VEGF 78 ± 7 83 ± 12 76 *An average between 4independent extract preparations. **An average between 6 independentextract preparations. ***% relative to the pre-SDR material.

The results show that preparing an extract contacting with PVP K25during the S/D removal step resulted in a similar and, with regards tosome growth factors, even superior (e.g. PDGF-AB and PDGF-BB) growthfactor recovery compared to contacting the lysate with dextran sulfateor heparin.

Advantageously, a platelet extract comprising PVP has no inhibitoryeffect on thrombin activity in-vitro as compared to heparin (shown inthe Examples above) and dextran sulfate at certain concentrations (datanot shown).

Example 15: The Effect of a Platelet Extract Prepared as Disclosed onSkin Healing in an In-Vivo Model

Tissue ischemia due to compromised blood flow is a major contributor tosurgical skin flap failure.

In this experiment, a modified McFarlane rat pedicle skin flap model wasused (McFarlane et al., Plast Reconst Surg (1965) 35:177) to evaluatethe ability of a platelet extract prepared as disclosed in promotingflap healing.

16 male Spargue-Dawley rats weighing 350-450 g (n=4 per treatment group)were used in this study.

Surgical Procedure:

The dorsal hair was removed one day prior to surgery to uncover theskin. On the day of surgery, animals received pre-operative analgesic inthe form of one dose of buprenorphine SQ injection (0.05 mg/Kg), andpre-operative antibiotics (Enrofloxacin: 10 mg/Kg). Anesthesia wasadministered by isoflurane inhalation (about 1-4%). The surgical sitewas prepared using chlorohexidine gluconate and 70% isopropyl alcohol. Athree-sided rectangular shaped dorsal full thickness flap in the size ofabout 10×3 cm was created. The flap margins were incised on three sides(cranially to caudally) leaving the flap attached along the caudal edge.The flap was elevated by blunt dissection and the exposed underlyingdorsal surface of the flap was progressively drip-coated with 1.5 ml offibrin sealant (prepared from a BAC2 component comprising a finalconcentration of 3 mg/ml human fibrinogen, and a final concentration of250 IU/ml human thrombin) with: i) an extract comprising PVP, ii) anextract comprising heparin or iii) Water for Injection (WFI; as controlI). See preparation of the extract and of the administered material andthe exact application procedure below. The flap was put back into itsanatomical place as the sealant was applied.

The skin flap was repositioned to its correct anatomic location,approximating the edges of the skin surrounding the flap and avoidingdead space by applying gentle pressure during the initial time periodrequired for the sealant to react and form a gel-like consistency. Theflap incision was then sutured back in its correct anatomic locationusing a 4-0 non-absorbable monofilament suture material in a consistentsimple interrupted pattern.

A control group (referred to as “control II”) underwent the same flapcreation procedure, but did not have any treatment applied to theunderlying dorsal surface prior to flap closure.

Extract Preparation:

Two platelet extract preparations were examined: one comprising PVP(prepared as in Example 10 PVP); and the other comprising heparin(prepared as in Example 14). Both extracts were then subjected to a stepof stabilization, pasteurization and removal of the stabilizers bydiafiltration against acetate-glycine buffer as follows:

One gram sucrose per gram of sample was slowly added into the extractmaterial while mixing (at about 22° C.) until the sucrose was completelydissolved. Then, the solution was warmed to 37±1° C. and 0.11 g glycineper g of extract material was slowly added into the solution whilemixing and adjusting the pH to 6.8-7.4 using 0.5N NaOH. pH adjustmentwas carried out until the glycine was completely dissolved. This wasfollowed by a gradual addition of 0.8 g sucrose per g extract materialwhile mixing at 37° C. until completely dissolved. Sucrose and glycinewere added into the solution to serve as stabilizers during thepasteurization step. The solution was then pasteurized by heat treatmentat 60° C. for 10 hours with constant mixing (50 RPM). In order totransfer the resulting viscous solution (which was formed as a result ofthe stabilizers addition) into a clean vessel, it was diluted withacetate-glycine buffer (20 mM sodium acetate, and 10 mM glycine at pH6.8-7.4) up to a total weight of 14,000-14,300 g (about 12,000 ml). Thestabilizers were removed from the solution by diafiltration againstacetate-glycine buffer (20 mM sodium acetate, 10 mM glycine, pH 6.8-7.4)using Centramate System with 2 Omega 10 kDa cassettes (Pall Corp, PortWashington, N.Y., USA). The diafiltration step was carried out asfollows: the sample was first concentrated to a volume of 1800 ml, anddialysis was carried out against a total volume of 10,800 mlacetate-glycine buffer (20 mM sodium acetate, 10 mM glycine, pH 6.8-7.4)by a gradual addition of the buffer and keeping the solution volume at1,800±200 ml. The dialyzed solution was then concentrated to 410-445 ml.

For stabilization, Mannitol was added into the solution at a finalconcentration of 2% w/w. In order to remove aggregated material, thesolution was filtered through 1.2 μm Sartopure PP2 filter and 0.45 μmSartopore 2 filter. Sterile filtration was carried out under asepticconditions using a 0.2 μm Sartopore 2 filter.

The obtained solution was then aliquoted into 4 ml portions intoautoclaved glass vials, lyophilized and sealed with autoclaved rubberstoppers under nitrogen atmosphere and in partial vacuum (0.6 Bar).

Preparation of BAC2 (Fibrinogen Comprising Component)+Extract:

The lyophilized extract's vial cap was aseptically opened and 1 ml ofsterile Water for Injection (WFI) was slowly added (without pipetting upor down or vortexing as to not create foaming) to the vial oflyophilized extract. The cap was aseptically replaced back on the vialand the vial was placed on a tube roller/rocker for approximately 5minutes at room temperature or until the extract powder became fullyreconstituted.

Next, 2 ml BAC2 (fibrinogen component of EVICEL® fibrin sealant: OmrixBiopharmaceuticals Ltd.;

comprising 60 mg fibrinogen/ml as in EVICEL®) was aseptically mixed with18 ml BAC2 dilution buffer (to make 6 mg fibrinogen/ml) containing 120mM sodium chloride, 10 mM tri-sodium citrate, 120 mM glycine, 95 mMarginine hydrochloride, 1 mM calcium chloride, pH-7.0-7.2. The vial wasgently agitated for at least 5 minutes. Then, 2 ml diluted BAC2 (6 mgfibrinogen/ml) was drawn into a syringe without a needle and mixed withthe above mentioned 1 ml rehydrated extract-heparin; extract-PVP or WFI.The vials were placed on a tube roller/rocker for approximately 5minutes at room temperature until use.

Preparation and Application of the Tested Article:

The yellow triple lumen catheter tip of the EVICEL® application devicewas cut at its base and replaced with a 16 G single lumen Venflon™Intravenous Catheter (without the needle) or other appropriately sizedcatheter. The vial connectors and the syringes were removed from thedevice. One of the 3 ml syringes was replaced with a 1 ml syringe, and 1ml of thrombin component (EVICEL fibrin sealant; OmrixBiopharmaceuticals Ltd.) was aseptically drawn up straight from the vialand into the 1 ml syringe. Three ml of BAC2+a platelet extractcomprising heparin; BAC2+a platelet extract comprising PVP; or BAC2+WFI(prepared as described above) were aseptically drawn up into the 3 mlsyringe.

The two syringes (one containing 1 ml thrombin; and the other containing3 ml of BAC2 and extract or WFI solution were placed in the bluebarreled syringe holder (from a new EVICEL® application kit) and theprovided plastic blue end connector was placed over the ends of bothplungers such that both thrombin and the tested solution could beadministered simultaneously. As mentioned above, a total volume of 1.5ml fibrin sealant with: i) an extract comprising PVP, ii) an extractcomprising heparin or iii) Water for Injection (WFI: as control 1) wasadministered in a volume ratio of 3 (fibrinogen comprising componentwith or without extract):1 (thrombin component) [1.125 ml:0.375 ml].

The growth factor concentration per ml (measured using the ELISA kitdescribed above) in the material administered to the rat was as shown inTable 16 (a volume of 1.5 ml was administered).

TABLE 16 Growth factor concentration in the tested extracts. Growthfactor (pg/ml) TGF-β1 PDGF-AB PDGF-BB VEGF EGF Extract-Heparin 2175024555 1323 1147 3777 Extract-PVP 211153 7417 1279 1844 3790

Evaluation: At 14 days after surgery the animals were anesthetized, andthen euthanized by exposure to CO₂. The adherence of a healthy(non-necrotic and soft tissue) area of the flap to the underlying tissuewas evaluated according to the following ranking (from worse to best):1—no flap adherence; 2—partial adherence; and 3—near normal or normalflap adherence.

The scoring/grade for the various treatment groups are shown in Table17.

TABLE 17 Adherence grade for normal appearing area of the skin flap(scale of 1-3) for the various treatment groups. Adherence grade fornormal appearing area of the skin flap (scale of 1-3) FS* + FS + aplatelet a platelet, extract extract comprising comprising AnimalControl II FS* alone heparin PVP 1 2 2 3 3 2 2 2 3 3 3 2 2 3 3 4 2 1 2 3AVERAGE 2 1.75 2.75 3 *FS - fibrin sealant.

The results show that administering fibrin sealant in combination with aplatelet extract comprising PVP had a similar positive effect inpromoting skin flap adherence as administering the fibrin sealant with aplatelet extract comprising heparin, both were superior to FS alone orControl II.

Once the macroscopic evaluation was completed, the skin flaps werecollected including about 0.5 cm of normal skin adjacent to the lateraledges. The abdominal and thoracic viscera were removed through ventralmidline incision. The collected tissue was placed into 10% neutralbuffered formalin. After adequate fixation, tissue sections were takenapproximately every 2 cm starting from the caudal end and designated asareas A-E, as shown in FIG. 6, perpendicular to the right side of theskin flap in such a manner that normal tissue and the skin flap wereincluded in each tissue section. The tissue sections were processed(infiltrated and embedded in paraffin) and the paraffin blocks were thensectioned using a microtome (5 micron). The sections were mounted onSuper Frost+™ slides, and assessed by histology andimmunohistochemistry.

Hematoxylin & Eosin staining: Paraffin embedded skin/wound-sectionslides were incubated at 60° C. for 30 minutes and deparaffinized bywashing the slides twice with xylene (100%) for 5 minutes, followed byrehydration in decreasing concentrations of ethanol in DDW (100-70%) for5 minutes in each concentration. The slides were stained withhematoxylin (ready for use solution) for 8 minutes, rinsed with water,immersed for a few seconds in 1% HCl/70% ethanol and then stained witheosin (0.5% in DDW) for 6 minutes. The sections were then washed with70% ethanol by 2 quick immersions. Thereafter, the slides weredehydrated by washing once with 95% ethanol for 5 minutes, twice withabsolute ethanol for 5 minutes, and twice with xylene (100%) for 3minutes and then mounted with Entellan mounting medium (MERCK DarmstadtGermany).

Immunohistochemistry staining: Paraffin embedded skin incision sectionslides were prepared as described above. The slides were incubated withblocking solution (10% normal serum) for 1 hour followed by incubationwith one of the following primary antibody: directed against keratin 6,keratin 1, keratin 14 (Covance), PCNA (Santa Cruz) overnight at 4° C.The next day, slides were washed with 0.05% Tween in PBS and incubatedwith the corresponding secondary biotin-conjugated antibody (VectorLabs) for 1 h. Detection was carried out using ABC Elite kit (VectorLabs), following manufacture instructions. Slides were then rinsed withwater, counter-stained with Mayer's hematoxylin for 30 seconds, and thenwashed with 70% ethanol by 2 quick immersions. Thereafter, the slideswere dehydrated by washing once with 95% ethanol for 5 minutes, twicewith absolute ethanol for 5 minutes, and twice with xylene (100%) for 10minutes, then mounted with Entellan mounting medium (MERCK DarmstadtGermany).

Analysis Criteria.

Epidermal hyperplasia: Hyperplastic response as measured by epidermalthickness was determined by H&E staining, where a sample was scored asone, when its epidermal thickness was observed in at least one field tocontain >6 nucleated layers. Samples where 6 or less layers wereobserved on the entire section were scored as zero.

Dermal hyperproliferation: Hyper-proliferative granulation tissue wasassessed utilizing PCNA staining of proliferating nuclei, where score 1was assigned when >10 nuclei per field (×40) were counted at the dermalincision area. Score 0 was assigned when 10 or less nuclei were counted.

Suprabasal keratin 6: Suprabasal keratin 6 was scored 1 when woundsdisplayed extensive distribution of K6 staining presented by brownstaining.

Suprabasal proliferation: Non healed wounds display proliferating cellsin several layers above the basal layer at the wound gap, observed byPCNA staining. When suprabasal proliferation was observed in at leastone area of the sample, it was scored as 1. Upon advanced healing,proliferation is observed only at the basal layer, which was scored as0. Taken together, for all markers, a score of zero represents a moreadvanced healing stage relative to score 1.

FIG. 7 shows representative stainings for the four tested markers fromthis study. Epidermal hyperplasia, dermal hyperproliferation, suprabasalkeratin 6 staining and suprabasal proliferation (left panel) were scoredas 1. Representative fields are presented for H&E staining (epidermalhyperplasia), PCNA staining (dermal and epidermal proliferation) andKeratin 6 staining. Yellow arrows in the epidermal hyperplasia panelportray epidermal thickness. Yellow arrows in the K6 panel indicated K6keratin distribution as presented in brown staining. Red arrowsdemonstrate PCNA positive nuclei of proliferating cells.

As shown in Table 18, flaps treated with FS+PEX-PVP showed more advancedhealing i.e. more animals scored 0 (for definition of scores 0 and 1,see “Methods”), than sham- or FS-treated flaps for 4 different healingmarkers. FS+PEX-PVP treated wounds displayed a significant reduction inseveral characteristics of active wounds which included reduction inepidermal hyperplasia, reduction of dermal fibroblast and epidermalkeratinocyte proliferation and diminished Keratin 6 staining. Reducedhyperplasia represents the thinning of the epidermis characteristic tonormal skin. Reduction of dermal fibroblast and suprabasal keratinocyteproliferation (shown by PCNA staining) marks mature matrix andremodeling of the dermis and epidermis, and reduced Keratin 6 staininglimited to a single cell layer at the basal epidermis marksnormalization of skin characteristics. In contrast, control (FS) treatedrat flaps displayed an early immature healing stage

TABLE 18 Comparison of healing markers in PEX-PVP vs. control treatmentsin a rat dorsal flap model. Area Sham FS Control FS + PEX-PVP EpidermalA 2/4 2/4 0/4 Hyperplasia C 2/4 3/4 0/4 Dermal C 2/4 2/4 1/4hyperproliferation D 3/4 3/4 2/4 Suprabasal A 2/4 3/4 0/4 proliferationSuprabasal A 1/4 2/4 0/4 Keratin 6 C 2/4 3/4 1/4

Example 16: Large Scale Process of Platelet Lysate Preparation fromPooled Washed Aphaeresis Platelets, Leukocyte Reduced (WAP), Treatedwith S/D, Incubated with PVP K12, Subjected to SDR Column at thePresence of PVP K12, Treated with Stabilizers, Concentrated withUltrafiltration/Diafiltration (UF/DF) System and Lyophilized

In this experiment, the effect of PVP K12 addition during S/D removal onthe recovery of growth factors was evaluated in a large scale process(see example 10). Lower molecular weight (LMW)-PVP, e.g. PVP K12, ismore suitable for formulation of parenteral drugs than higher molecularweight PVPs (PVP 25), since the first permits rapid renal eliminationwithout storage. Also, in some countries in Europe, e.g. Germany andAustria, only such low-molecular PVP types with a K-value of up to 18are approved for injection. Platelet lysate samples were prepared using1958 g pooled washed apheresis platelet leukocyte reduced (WAP) obtainedfrom 10 bags. 214 ml acetate-glycine final concentration of 20 mM sodiumacetate, 10 mM glycine; at pH 6.8-7.4 (AGA buffer) and Human SerumAlbumin (HSA; Talecris USA, final concentration of 0.2% v/v) were addedinto the pooled WAP. In the next step, S/D treatment was carried out byslowly adding Triton X-100 and TnBP 1% and 0.3% (v/v) finalconcentration, respectively) while mixing at 30 RPM. In order to avoidpotential sub-optimal viral inactivation due to the possible presence ofparticulate matter, the S/D treatment was split into two steps. First,the sample was continuously stirred for 30 minutes, centrifuged at5016×g for 10 minutes at 23-27° C. and filtered through a 0.45 μm filter(Sartopore 2, Sartorius Stedim Biotech S.A., Aubagne, France). Then, thefiltered material was poured into a stainless steel pot immersed in awater bath adjusted to 25° C. and mixed at 30 RPM for additional 2 hoursto continue the viral inactivation process in a clear solution. PVP K12(Polyvinylpyrrolidone K12 with a K-Value of 12 and an average molecularweight of 3500 Da, Cat. 276142500 Acros organics. Germany) was added tothe S/D-treated lysate to a final concentration of 0.3% (w/w) i.e. 0.857mM) and incubated for 20 minutes at 25° C. while stirring at 30 RPM. Thesample was filtered using 5 μm Sartopore PP2 filter to removeparticulate matter.

Next, S/D removal was carried out on a XK50 liquid chromatography columnpacked with 300 ml SDR HyperD solvent-detergent removal chromatographyresin (Pall Corp) in conjunction with a peristaltic pump and a UA-6UV/VIS detector+Type 11 recorder (ISCO, NE, USA). The column wasequilibrated with 900 ml of AGA buffer (as above) containing 0.3% (0.857mM) PVP K12. 1800 ml of S/D- and PVP-treated platelet lysate (whichcontained 1620 ml of platelet material) were loaded onto the columnfollowed by washing the column with 600 ml AGA buffer (as above)containing 0.3% (0.857 mM) PVP K12. This was followed by washing with600 ml of AGA buffer containing 12.5% ethanol, 1M NaCl and 0.3% (0.857mM) PVP K12. Flow-through and washing fractions were collected andcombined for growth factor recovery calculations.

A total volume of 3000 ml was collected from the column. The collectedmaterial was filtered consecutively with 3 and 1.2 μm Sartopure PP2filters and 0.45 μm Sartopore 2 filter (Sartorius Stedim Biotech S.A.,Aubagne, France).

PDGF-AB, PDGF-BB, VEGF, TGF-031, EGF and bFGF recoveries were calculatedas described above and are shown in Table 19 below.

TABLE 19 Comparison between recoveries of growth factors in a plateletlysate following S/D removal step in the presence of (PVP K12 or PVPK25). Growth factor Recovery (%)* Lysate-PVP K25 (0.1 [0.03 mM]-Lysate-PVP K12 0.5%[0.17 nM])** (0.3%(0.857 mM)) TGF-b1 83 65 PDGF-AB 7375 PDGF-BB 87 73 EGF 59 60 bFGF 61 74 VEGF 76 96 *Compared to pre-SDstep **For detailed conditions, see example 10.

The results presented in Table 19 show that the high recoveries ofgrowth factors from the SDR column in the presence of PVP K25 aremaintained when using lower molecular weight PVP e.g. PVP K12.

Next, the filtered material was poured into a stainless steel potimmersed in a water bath adjusted to 25° C. and mixed at 30 RPM. Inpreparation for viral heat inactivation, a stabilizing procedure wascarried out in three steps. First, 100% sucrose (w/w) was added to thematerial in small portions. Once the sucrose was completely dissolved,the solution was warmed to 37° C., and 10% glycine (w/w) was slowlyadded at pH 6.9-7.1. In the last step of stabilizer addition, 80%sucrose (w/w) was added and further mixed until completely dissolved.Then, the heat viral inactivation step was carried out at 59.5-60.5° C.for 10 hours while stirring at 20 RPM.

At the end of the viral inactivation step, 40% acetate-glycine buffer(w/w) was added to the solution while mixing at 37° C. at 30 RPM. Thesolution was then filtered using 3 μm Sartopure PP2 filter (SartoriusStedim Biotech S.A., Aubagne, France). Next, concentration andstabilizers removal procedures were carried out using Centramate™ UFHolder system installed with 4 Omega™ Centramate™ 10 kDa cassettes (Pallcorp) in 3 steps. First, the material was concentrated to a final volumeof 1800 ml. In the second step, 10800 ml of acetate-glycine buffer weregradually added while maintaining the volume of the solution between1600 to 2000 ml in order to replace the stabilizing buffer with a freshbuffer. Third, at the end of the dialysis step, the sample wasconcentrated to a final volume of 560 ml.

Mannitol (Sigma-Aldrich, St. Louis, Mo., USA) was added to the materialto a final concentration of 2% (w/w). Next, the material was filteredusing consecutively 1.2 μm Sartopure 300 filter, 0.45 μm Sartopore 2filter and 0.2 μm Sartopore 2 300 filter (Sartorius Stedim Biotech S.A.,Aubagne, France). 80 glass vials were then filled with 4 ml of thesolution and freeze-dried using Epsilon 2-8D lyophilizer (Martin ChristGmbH).

The concentrations of Triton X-100 and TnBP were measured prior to(pre-SDR material) and after the S/D removal step (post-SDR material)and at the end of the production process (Final). Triton X-100 wasdetermined by reversed phase HPLC with a U.V. detector, and TnBP wasdetermined by capillary gas chromatography using a Flame IonizationDetector.

The results are shown in Table 20 below.

TABLE 20 S/D concentration in the lysate prior to, following S/D removaland at the end of the production process according to the conditionselaborated above. Material S/D ppm pre-SDR material Triton X-100 8,795TnBP 2,526 post-SDR material Triton X-100 170 TnBP <0.3 Final TritonX-100 <5 TnBP <0.3

The results show that carrying out S/D removal in the presence of PVPK12 at a concentration of 0.3% (0.857 mM), which was found to beefficient for growth factor recovery, resulted in efficient removal ofTnBP and almost complete removal of Triton X-100 in the post-SDRintermediate (less than 2% of the starting material remained). Thetraces of Triton X-100 that remained in the material after the columnwere removed in the downstream process.

Example 17: The Effect of Contacting a Platelet Lysate with PVP withDifferent Molecular Weights at Different Concentrations During Loadingon S/D Removal Column on Growth Factor Recovery

The aim of this example was to investigate the effect of PVPs withdifferent molecular weights at different concentrations on growthfactors recovery following S/D removal.

In this example different S/D removal conditions were tested such as theaddition of four different molecular weight PVPs prior to and during theHIC S/D removal step. The recovery of the following growth factors wasexamined: PDGF-AB, bFGF, VEGF, and EGF as determined by ELISA.

PVP K12 (Polyvinylpyrrolidone K12 having a K-Value of 12 and an averagemolecular weight of 3500 Da, Cat. 276142500 Acros organics, Germany),PVP K17 (Polyvinylpyrrolidone K16-18 having an average K-Value of 17 andan average molecular weight of 8000 Da, Cat. 22746500 Acros organics,Germany), PVP K25 and PVP K30 were used in this example.

The lysates were prepared and loaded onto a SDR column as elaborated inExample 5 above. All S/D removal conditions are elaborated in Table 21.Prior to loading, equilibration was carried out using the buffer of thelysate to be loaded. All loaded samples were also incubated with PVPprior to SDR application, in the manner described above. All fractionsobtained from the column after loading, and after washing with the washbuffer were collected, combined, and the growth factor recovery wascalculated.

Growth factor recovery results (calculated as explained above) are shownin Table 22.

TABLE 21 A detailed description of samples and conditions used duringthe S/D removal step. Treatment Sample loaded Washing buffer 1 S/Dtreated lysate + 0.3 mM PVP K12 AGA + 0.3 mM PVP K12 2 S/D treatedlysate + 0.4 mM PVP K12 AGA + 0.4 mM PVP K12 3 S/D treated lysate + 0.9mM PVP K12 AGA + 0.9 mM PVP K12 4 S/D treated lysate + 1.7 mM PVP K12AGA + 1.7 mM PVP K12 5 S/D treated lysate + 0.2 mM PVP K17 AGA + 0.2 mMPVP K17 6 S/D treated lysate + 0.3 mM PVP K17 AGA + 0.3 mM PVP K17 7 S/Dtreated lysate + 0.8 mM PVP K17 AGA + 0.8 mM PVP K17 8 S/D treatedlysate + 1.5 mM PVP K17 AGA + 1.5 mM PVP K17 9 S/D treated lysate + 0.2mM PVP K25 AGA + 0.2 mM PVP K25 10 S/D treated lysate + 0.3 mM PVP K25AGA + 0.3 mM PVP K25 11 S/D treated lysate + 0.4 mM PVP K25 AGA + 0.4 mMPVP K25 12 S/D treated lysate + 0.1 mM PVP K30 AGA + 0.1 mM PVP K30 13S/D treated lysate + 0.3 mM PVP K30 AGA + 0.3 mM PVP K30 14 S/D treatedlysate + 0.4 mM PVP K30 AGA + 0.4 mM PVP K30

TABLE 22 Recovery of growth factors in a platelet lysate following S/Dremoval step at the presence of different MW PVPs (conditions in Table21). Growth Factor Recovery (%)* PDGF- Treatment Details AB bFGF EGFVEGF 1 0.3 mM PVP K12 17 28 62 78 2 0.4 mM PVP K12 18 31 68 88 3 0.9 mMPVP K12 38 83 67 102 4 1.7 mM PVP K12 71 96 77 108 5 0.2 mM PVP K17 4152 64 96 6 0.3 mM PVP K17 42 62 84 103 7 0.8 mM PVP K17 77 95 71 111 81.5 mM PVP K17 89 123 72 106 9 0.2 mM PVP K25 51 68 61 98 10 0.3 mM PVPK25 55 83 66 98 11 0.4 mM PVP K25 53 102 68 95 12 0.1 mM PVP K30 40 4561 83 13 0.3 mM PVP K30 62 91 67 104 14 0.4 mM PVP K30 55 89 67 104 *%relative to the pre-SDR column material.

The results show that increasing the concentration of each PVPcorrelates within creasing growth factors recovery, especially forPDGF-AB and bFGF. Also, increased growth factor recovery is observedwith increased molecular weight of PVP at identical molar concentration:For example, using PVP K12, K17, K25 or K30 at 0.3 mM, resulted inPDGF-AB recovery of 17, 42, 55 and 62%, respectively.

In order to evaluate the efficacy of S/D (Triton X-100 and TnBP)removal, the concentrations of Triton X-100 and TnBP were measured prior(pre-SDR material) to and following (post-SDR material) the S/D removalstep. Triton X-100 concentration was determined by reversed phase HPLCwith a U.V. detector, and TnBP was determined by capillary gaschromatography using a Flame Ionization Detector.

TABLE 23 S/D concentration in the lysate prior to and following S/Dremoval according to the conditions elaborated above. Treatment Detailsppm Pre-SDR material for treatments Triton X-100 8228 1, 5, 6, 9, 10TnBP 2750 Pre-SDR material for treatments Triton X-100 7936 2, 3, 11,12, 14 TnBP 2168 Pre-SDR material for treatments Triton X-100 8095 4, 7,8 TnBP 2374 Pre-SDR material for treatment 13 Triton X-100 7350 TnBP2181 1 0.3 mM PVP post-SDR Triton X-100 <5 K12 material TnBP <0.3 2 0.4mM PVP post-SDR Triton X-100 13 K12 material TnBP <0.3 3 0.9 mM PVPpost-SDR Triton X-100 52 K12 material TnBP <0.3 4 1.7 mM PVP post-SDRTriton X-100 173 K12 material TnBP <0.3 5 0.2 mM PVP post-SDR TritonX-100 <5 K17 material TnBP <0.3 6 0.3 mM PVP post-SDR Triton X-100 18K17 material TnBP <0.3 7 0.8 mM PVP post-SDR Triton X-100 39 K17material TnBP <0.3 8 1.5 mM PVP post-SDR Triton X-100 86 K17 materialTnBP <0.3 9 0.2 mM PVP post-SDR Triton X-100 <5 K25 material TnBP <0.310 0.3 mM PVP post-SDR Triton X-100 7 K25 material TnBP <0.3 11 0.4 mMPVP post-SDR Triton X-100 82 K25 material TnBP <0.3 12 0.1 mM PVPpost-SDR Triton X-100 <5 K30 material TnBP <0.3 13 0.3 mM PVP post-SDRTriton X-100 <5 K30 material TnBP <0.3 14 0.4 mM PVP post-SDR TritonX-100 56 K30 material TnBP <0.3

The results show an efficient removal of S/D during the S/D removal stepby HIC column. While all TnBP is removed, some residuals of Triton X-100are found in post-SDR material when using PVP in concentrations of 0.4mM and above. It was shown in Example 16 Table 20 above that residualTriton X-100 in the intermediate material, even slightly higher than theconcentrations presented here, was removed downstream to the presentedproduction process.

Example 18: The Effect of Using PVP in Different Stages of S/D Removalon Growth Factor Recovery

In order to identify at which stage in the multistep process of PVPapplication to the S/D removal column PVP's have the greatest effect onrecovery of growth factors, different combinations of PVP addition tothe three S/D removal steps were tested.

In this example the recovery of GFs during S/D removal step was testedwith the addition of 0.5% (w/w) (0.63 mM) PVP K17 to the plateletlysate, to the equilibration buffer and to the washing buffer atdifferent combinations as shown in Table 24.

The recovery of the following growth factors was examined: PDGF-AB,bFGF, VEGF, and EGF using ELISA. The lysates were prepared and loadedonto a SDR column as elaborated in Example 5, in larger scale processabove, with the exception of using equilibration and washing buffers ineach treatment as shown in Table 24. Flow through and washing fractionso were collected and combined and the growth factor recovery wascalculated.

Growth factor recovery results (calculated as explained above) are shownin Table 25.

TABLE 24 A detailed description of samples and conditions used duringthe S/D removal step. Treatment  Sample loaded Equilibration bufferWashing buffer 1 S/D treated lysate+ AGA AGA− 2 S/D treated lysate+ AGA+AGA− 3 S/D treated lysate+ AGA− AGA+ 4 S/D treated lysate+ AGA+ AGA+ 5S/D treated lysate− AGA+ AGA 6 S/D treated lysate− AGA+ AGA+ 7 S/Dtreated lysate− AGA− AGA+ +PVP K17 was added at 0.5% (0.63 mM). −Noaddition of PVP K17. *% relative to the pre-SDR material.

TABLE 25 Recovery of growth factors in a platelet lysate following S/Dremoval step according to the samples and conditions in Table 24.Results are sorted in a descending order according to PDGF-AB recovery.0.5% PVP K17 Growth factor Recovery (%)* Treatment presence PDGF-AB bFGFEGF VEGF 2 S/D treated 74 96 77 133 LysateEquilibration buffer 4 S/Dtreated 69 100 76 123 LysateEquilibration buffer Washing buffer 6Equilibration buffer 55 75 71 115 Washing buffer 5 Equilibration buffer38 68 74 105 1 S/D treated Lysate 36 59 65 117 3 S/D treated Lysate 2957 67 111 Washing buffer 7 Washing buffer 21 36 61 103 *% relative topre-SDR material.

In three samples, 0.5% PVP K17 was added only to one step: to theplatelet lysate (treatment #1), to the equilibration buffer (treatment#5) or to the washing buffer (treatment #7).

Addition of PVP K17 only to the platelet lysate or to the equilibrationbuffer resulted in similarly low growth factor recovery, whereasaddition of PVP K7 only to the washing buffer resulted in the lowestimprovement in GFs recovery, compared with the two other treatments.

Three additional treatments (treatment #2, #3 and #6) includedcombinations between two of the three conditions while a treatment #4included a combination of all three conditions.

The results show that the most effective single step in increasing GFsrecovery from the HIC column was addition of PVP to the equilibrationbuffer. Moreover, the highest GFs recovery was achieved using acombination of PVP in the equilibration buffer with addition of PVP tothe lysate.

In order to evaluate the efficacy of S/D (Triton X-100 and TnBP)removal, the concentrations of Triton X-100 and TnBP were measured prior(pre-SDR material) to and following (post-SDR material) the S/D removalstep. Triton X-100 was determined by reversed phase HPLC with a U.V.detector, and TnBP was determined by capillary gas chromatography usinga Flame Ionization Detector.

TABLE 26 S/D concentration in the lysate prior to and following S/Dremoval according to the conditions elaborated above. 0.5% (0.63 mM)Treat- PVP K17 ment presence Material S/D ppm pre-SDR material fortreatments 1-4 Triton X-100 8601 TnBP 2516 pre-SDR material fortreatments 5-7 Triton X-100 8322 TnBP 2564 2 Lysate post-SDR materialTriton X-100 101 Equilibration TnBP <0.3 buffer 4 Lysate post-SDRmaterial Triton X-100 73 Equilibration TnBP <0.3 buffer Washing buffer 6Equilibration post-SDR material Triton X-100 7 buffer TnBP <0.3 Washingbuffer 5 Equilibration post-SDR material Triton X-100 30 buffer TnBP<0.3 1 Lysate post-SDR material Triton X-100 <5 TnBP <0.3 3 Lysatepost-SDR material Triton X-100 7 Washing buffer TnBP <0.3 7 Washingbuffer post-SDR material Triton X-100 <5 TnBP <0.3

The results show an efficient removal of S/D during the S/D removal stepby HIC column. While all TnBP was removed in all treatments, someresiduals of Triton X-100 were found in post-SDR material when using PVPboth in the equilibration buffer and in the platelet lysate. However, itwas shown in example 16 above that higher concentration of Triton X-100in the intermediate material than the concentrations presented here wereremoved in the downstream process.

Addition of PVP to the equilibration buffer as well as to the washingbuffer resulted in high recovery of growth factors, withoutsignificantly compromising the efficacy of triton X-100 removal by theHIC column.

Example 19: The Effect of Admixing a Platelet Lysate with HPMC Prior toS/D Removal on Growth Factor Recovery

In order to test if other amphiphilic polymers can be used to improvethe recovery of growth factors during the S/D removal step by HICcolumn, Hydroxy Propyl Methyl Cellulose (HPMC) was explored in thefollowing example.

HPMC is a natural multifunctional carbohydrate polymer currently widelyused as an excipient and controlled-delivery component in oralmedicaments and as an emulsifier in food industry.

In this example, two different concentrations of HPMC (10 KDa, Cat.423238 Sigma-Aldrich, USA) were tested prior to and during HIC S/Dremoval step. S/D removal step without additives was tested as control.

The lysates were prepared and loaded onto a SDR column as elaborated inExample 5 above. In order to investigate the effect of HPMC alone(without NaCl and EtOH), in this example only 1 buffer with volume of 15ml was used for washing. All the conditions are elaborated in Table 27.Prior to loading, equilibration was carried out using the same buffer asthe one used in the lysate loaded. Flow through and washings werecollected, combined, and the growth factor recovery was calculated.Growth factor recovery results (calculated as explained above) are shownin Table 28 All loaded samples that comprised HPMC were incubated withHPMC in the manner discussed above.

The recovery of the following growth factors was examined: PDGF-AB,b(basic) FGF, VEGF, and EGF using ELISA.

TABLE 27 A detailed description of samples and conditions used duringthe S/D removal step. Treatment Sample loaded Washing Buffer 1 S/Dtreated lysate AGA 2 S/D treated lysate + AGA + 0.1% (0.1 mM) HPMC 0.1%(0.1 mM) HPMC 3 S/D treated lysate + AGA + 0.3% (0.3 mM) HPMC 0.3% (0.3mM) HPMC

TABLE 28 Recovery of various growth factors in a platelet extractfollowing S/D removal step according to the samples and conditions inTable 27. Growth factor Recovery (%)* Treatment PDGF-AB bFGF EGF VEGF 17 13 44 48 2 22 25 55 66 3 28 39 63 89 *% relative to pre-SDR column.

The results show that contacting an S/D-treated platelet lysate withHPMC prior to and during S/D removal by an SDR column improved therecovery of growth factors from an SDR column.

Also, the results show that higher concentration of HPMC during the S/Dremoval step resulted in increased recovery of growth factors.

The concentrations of Triton X-100 and TnBP were measured prior (pre-SDRmaterial) to and following (post-SDR material) the S/D removal step.Triton X-100 was determined by reversed phase HPLC with a U.V. detector,and TnBP was determined by capillary gas chromatography using a FlameIonization Detector.

The results are shown in Table 29 below.

TABLE 29 S/D concentration in the lysate prior to and following S/Dremoval according to the conditions elaborated above. Treatment MaterialS/D ppm 1 pre-SDR material Triton X-100 8322 TnBP 2564 post-SDR materialTriton X-100 <5 TnBP <0.3 2 pre-SDR material Triton X-100 8492 TnBP 2555post-SDR material Triton X-100 <5 TnBP <0.3 3 pre-SDR material TritonX-100 8297 TnBP 2573 post-SDR material Triton X-100 <5 TnBP <0.3

The results show that carrying out an S/D material removal in thepresence of HPMC in a concentration of up to 0.3%, which was found to beefficient for growth factor recovery, did not affect the S/D removalperformance of the column.

TABLE 30 PVP and HPMC concentrations and MW. MW average Conc. Conc. PVPK-Value (Dalton) (% w/w) (mM) K12 10.2-13.8 3500 0.5 1.43 K17 16.0-18.08000 0.5 0.63 K25 22.5-27.0 30000 0.5 0.17 K25 22.5-27.0 30000 1 0.3 K3027.0-32.4 40000 0.5 0.13 HPMC — 10000 0.5 0.5

Example 20: The Ratio Between Several Growth Factors in a PlateletExtract Prepared from WAP, Treated with S/D, Contacted with PVP K12, andSubjected to S/D Removal

The following Example shows the ratio between several growth factors ina platelet extract prepared as disclosed, and examines whether theobtained ratio is comparable to that in the starting material, and tothat in the lysate before loading the sample onto the chromatographyresin (the pre-SDR material). The pre- and post-SDR material (orextract) were prepared as described in Example 16.

The levels of TGF-β1, VEGF, bFGF, and PDGF-AB were measured in all threetested materials (WAP starting material, pre-SDR material, and post-SDRmaterial) using the specific commercial ELISA kit described above, andthe ratios between PDGF-AB/TGF-β1; PDGF-AB/VEGF; TGF-β1/bFGF; andVEGF/bFGF were calculated. The growth factors levels and ratios areshown in Table 31 and 32, respectively, below.

TABLE 31 Levels of TGF-β1, VEGF, bFGF, and PDGF-AB in WAP startingmaterial, pre-SDR material, and post-SDR material. Growth factor level(ng) Tested material TGF-β1 VEGF bFGF PDGF-AB WAP starting material318740 1368 139 151810 Pre-SDR material 462757 1226 256 159791 Post-SDRmaterial 299010 1178 189 120126

TABLE 32 Calculated growth factors ratio in WAP starting material,pre-SDR material, and post-SDR material. Tested PDGF-AB/ PDGF-AB/TGF-β1/ VEGF/ material TGF-β1 VEGF bFGF bFGF WAP starting 0.48 111 22889.8 material pre-SDR 0.34 130 1810 4.8 material post-SDR 0.40 102 15826.2 material

The results show that a platelet lysate contacted with PVP K2 incombination with ethanol and NaCl during a S/D removal step results inan extract having PDGF-AB/TGF-β1; PDGF-AB/VEGF; TGF-β1/bFGF; andVEGF/bFGF ratios which are comparable to the ratios in the startingmaterial and in the material prior to S/D removal. It can be concludedthat carrying out an S/D removal as disclosed results in a plateletextract comprising a proportion of factors that is comparable to thematerial before S/D removal.

What is claimed is:
 1. A method for preparing a viral-safe biologicalliquid mixture composition from a biological source, the methodcomprising the following steps: providing the source as a clearsolution; providing a non-toxic amphiphilic polymer; treating the sourcewith a solvent detergent (S/D) to allow viral inactivation; treating thesource with the amphiphilic polymer at a final concentration of 0.01 to0.9 mM wherein the clarity of the solution is maintained; removing theS/D by contacting the treated source with an hydrophobic interactionchromatography (HIC) resin; and collecting a material comprising anunbound fraction from HIC; wherein the method comprises at least onemore orthogonal viral inactivation treatment, thereby obtaining theviral-safe biological liquid mixture composition.
 2. The methodaccording to claim 1, wherein removing the S/D omits a further step ofoil extraction.
 3. The method according to claim 1, wherein the HICresin is packed in a column.
 4. The method according to claim 1, whereinthe source comprises cells, cell particles and/or cell organelles. 5.The method according to claim 1, wherein the source is a blood buffycoat.
 6. The method according to claim 1, wherein the source comprisesplatelets.
 7. The method according to claim 1, wherein the source is aplatelet-enriched fraction pooled from multiple donors.
 8. The methodaccording to claim 1, wherein the amphiphilic polymer is a hydrocarbonbased surfactant.
 9. The method according to claim 1, wherein theamphiphilic polymer is selected from the group consisting ofpolyvinylpyrrolidone (PVP), hydroxy propylmethylcellulose (HPMC), and acombination thereof.
 10. The method according to claim 9, wherein thesource is contacted first with the S/D and then with the PVP and whereinthe PVP concentration in the S/D treated source is in the range of about0.01 to 0.3 mM.
 11. The method according to claim 9, wherein the sourceis contacted first with the S/D and then with the PVP and wherein thePVP concentration in the S/D treated source is in the range of about0.025 to 0.3 mM.
 12. The method according to claim 9, wherein the sourceis contacted first with the S/D and then with the HPMC and wherein theHPMC concentration in the S/D treated source is in the range of about0.01 to 0.3 mM.
 13. The method according to claim 1, wherein the sourceis contacted first with the S/D and then with the amphiphilic polymer.14. The method according to claim 1, wherein the method furthercomprises the steps of: washing the resin with a solution comprising anorganic solvent and/or a salt; collecting a fraction obtained followingthe washing step and combining with the unbound fraction.
 15. The methodaccording to claim 14, wherein the organic solvent is ethanol.
 16. Themethod according to claim 14, wherein the salt is NaCL.
 17. The methodaccording to claim 1, wherein the at least one more orthogonal viralinactivation treatment comprises heat inactivation.
 18. The methodaccording to claim 1, further comprising a step of concentrating thematerial.
 19. The method according to claim 1, further comprising a stepof drying the material, thereby resulting in a viral-safe biological drymixture.
 20. A method for preparing a biological liquid mixturecomposition from a biological source, the method comprising thefollowing steps: providing the source as a clear solution; providing PVPand/or HPMC; treating the source with a solvent detergent (S/D) to allowviral inactivation; treating the source with the PVP and/or HPMC at afinal concentration of 0.01 to 0.9 mM wherein the clarity of thesolution is maintained; removing the S/D by contacting the treatedsource with an hydrophobic interaction chromatography (HIC) resin; andcollecting a material comprising an unbound fraction from HIC.
 21. Amethod for removing solvent-detergent (S/D) from a biological sourcecomprising S/D, the method comprises the steps of: providing the sourceas a clear solution; providing an amphiphilic polymer; treating thesource with S/D and with the amphiphilic polymer at a finalconcentration of 0.01 to 0.9 mM wherein the clarity of the solution ismaintained; removing the S/D from the biological source by contactingthe treated source with an hydrophobic interaction chromatography (HIC)resin; and collecting a material comprising an unbound fraction fromHIC.
 22. The method according to claim 21, wherein removing the S/Domits a further step of oil extraction.